US20170334951A1 - Immunogenic wt-1 peptides and methods of use thereof - Google Patents

Immunogenic wt-1 peptides and methods of use thereof Download PDF

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US20170334951A1
US20170334951A1 US15/608,964 US201715608964A US2017334951A1 US 20170334951 A1 US20170334951 A1 US 20170334951A1 US 201715608964 A US201715608964 A US 201715608964A US 2017334951 A1 US2017334951 A1 US 2017334951A1
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peptide
cancer
peptides
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US10100087B2 (en
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Richard J. O'Reilly
Ekaterina Doubrovina
Annamalai Selvakumar
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Memorial Sloan Kettering Cancer Center
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Memorial Sloan Kettering Cancer Center
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001152—Transcription factors, e.g. SOX or c-MYC
    • A61K39/001153—Wilms tumor 1 [WT1]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464452—Transcription factors, e.g. SOX or c-MYC
    • A61K39/464453—Wilms tumor 1 [WT1]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes

Definitions

  • This invention provides peptides, compositions and vaccines comprising same, and methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer, comprising administering same.
  • Wilms tumor a pediatric nephroblastoma that occurs with a frequency of 1 in 10,000 births, has been the subject of intense clinical and basic research for several years.
  • the tumor is embryonic in origin; it is detected in children usually during the first 5 years of life and can occur unilaterally or bilaterally.
  • a WT arises when condensed metanephric mesenchymal cells of the developing kidney fail to properly differentiate.
  • WT1 Wilms tumor 1
  • WT1 Wilms tumor protein I
  • WT1 is a zinc finger transcription factor expressed during normal ontogenesis such as in fetal kidney, testis and ovary. In adults, WT1 expression is limited to low levels on hematopoietic stem cells, myoepithelial progenitor cells, renal podocytes and some cells in testis and ovary. Recent demonstration that WT1 is over expressed in several types of leukemia suggested that WT1 would be an attractive target for immunotherapy for various cancers.
  • This invention provides peptides, compositions, and immunogenic compositions such as vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer, comprising administering immunogenic peptides.
  • the present invention provides an isolated WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:1-160, 162-185, 190, 191 and 193.
  • the present invention provides an isolated HLA class I binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 190, 191 and 193.
  • the present invention provides an isolated HLA class II binding WT1 peptide having an amino acid (AA) sequence consisting of any one of
  • the present invention provides an isolated WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 1-160, 162-185, 190, 191 and 193, or a fragment of any of the foregoing.
  • AA amino acid
  • the present invention provides an isolated HLA class I binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 190, 191 and 193.
  • the present invention provides an isolated HLA class II binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:149, 156, 173, 174 and 180.
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) a peptide selected from SEQ ID NO:1-160, 162-185, 190, 191 and 193.
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class I binding peptide selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183.
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class II binding peptide selected from SEQ ID NO:149, 156, 173,
  • the present invention provides a vaccine comprising one or more peptides of SEQ ID NO:1-160, 162-185, 190, 191 and 193.
  • the present invention provides a vaccine comprising one or more HLA class I binding peptides selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183.
  • the present invention provides a vaccine comprising one or more HLA class II binding peptides selected from SEQ ID NO:149, 156, 173, 174 and 180.
  • the present invention provides a vaccine comprising one or more HLA class I binding peptides selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, and one or more HLA class II binding peptides selected from SEQ ID NO:149, 156, 173, 174 and 180.
  • the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.
  • the present invention provides a method of inducing formation and proliferation of a WT1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of a WT1 protein-specific CTL.
  • This method can be conducted in vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect.
  • the present invention provides a method of inducing formation and proliferation of (a) a WT1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT1 protein; or a combination thereof.
  • This method can be conducted in vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect.
  • the present invention provides a method of inducing an anti-cancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby inducing an anti-mesothelioma immune response in a subject.
  • the fragment of a WT1 protein consists of a peptide or comprises a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 and 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.
  • the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby treating a subject with a mesothelioma.
  • the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.
  • the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject.
  • the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.
  • the cancer is a WT1-expressing cancer.
  • the WT1-expressing cancer is an acute myelogenous leukemia (AML).
  • the WT1-expressing cancer is associated with a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • the WT1-expressing cancer is an MDS.
  • the WT1-expressing cancer is a non-small cell lung cancer (NSCLC).
  • the WT1-expressing cancer is a Wilms' tumor.
  • the WT1-expressing cancer is a leukemia.
  • the WT1-expressing cancer is a hematological cancer.
  • the WT1-expressing cancer is a lymphoma.
  • the WT1-expressing cancer is a desmoplastic small round cell tumor.
  • the WT1-expressing cancer is a mesothelioma.
  • the WT1-expressing cancer is a malignant mesothelioma.
  • the WT1-expressing cancer is a gastric cancer.
  • the WT1-expressing cancer is a colon cancer.
  • the WT1-expressing cancer is a lung cancer.
  • the WT1-expressing cancer is abreast cancer.
  • the WT1-expressing cancer is a germ cell tumor.
  • the WT1-expressing cancer is an ovarian cancer.
  • the WT1-expressing cancer is a uterine cancer. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a liver cancer. In another embodiment, the WT1-expressing cancer is a renal cancer. In another embodiment, the WT1-expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT1-expressing cancer is a sarcoma. In another embodiment, the WT1-expressing cancer is any other carcinoma or sarcoma.
  • the WT1-expressing cancer is a solid tumor.
  • the solid tumor is associated with a WT1-expressing cancer.
  • the solid tumor is associated with a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • NSCLC non-small cell lung cancer
  • the solid tumor is associated with a lung cancer.
  • the solid tumor is associated with a breast cancer.
  • the solid tumor is associated with a colorectal cancer.
  • the solid tumor is associated with a prostate cancer.
  • the solid tumor is associated with an ovarian cancer.
  • the solid tumor is associated with a renal cancer.
  • the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
  • the present invention provides a composition comprising an isolated peptide of the invention in combination with at least 1 additional WT1 peptide.
  • a composition comprising at least 2 different isolated peptides of the present invention is provided.
  • a composition comprising at least 3 or at least 4 different isolated peptides of the present invention is provided.
  • Each possibility represents a separate embodiment of the present invention.
  • the composition of the present invention is a vaccine.
  • the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a peptide or composition of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a peptide or composition of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.
  • the present invention provides a method of inducing formation and proliferation of a WT1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of a WT1 protein-specific CTL.
  • the present invention provides a method of inducing formation and proliferation of (a) a WT1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT1 protein; or a combination thereof.
  • the invention is directed to a peptide of the invention with at least one amino acid change that increases the affinity of the peptide for binding to a HLA molecule.
  • FIG. 2 A-E depicts the strategy for the generation of the total pool of overlapping pentadecapeptides spanning the whole sequence of the WT1 protein and epitope mapping;
  • FIG. 3 A-D shows that the combined HLA class I and II restricted WT1 specific T cell response to the same immunodominant peptide sequence derived from WT1 protein in the WT1 CTL after 40 days of co-culture with the WT1 total pool of overlapping 15-mers loaded on autologous CAMs;
  • FIG. 4A-F depicts schema of WT1
  • FIG. 5 depicts results using mixed A0201 epitopes loaded on A0201-AAPC in 8 normal A0201+ donors.
  • This invention provides immunogenic peptides, and compositions and vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer, comprising administering one or more immunogenic peptides.
  • This invention provides WT1 peptides and methods of treating, reducing the incidence of, and inducing immune responses against a WT1-expressing cancer, comprising immunogenic peptides.
  • the WT1 molecule from which the peptides of the present invention are derived has, in another embodiment, the sequence:
  • the WT1 sequence is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
  • the WT1 molecule has the sequence:
  • the WT1 molecule has the sequence:
  • the WT1 protein has the sequence set forth in GenBank Accession # NM_024426. In other embodiments, the WT1 protein has or comprises one of the sequences set forth in one of the following sequence entries: NM_024425, NM_024424, NM_000378, 595530, D13624, D12496, D 12497, or X77549. In another embodiment, the WT1 protein has any other WT1 sequence known in the art.
  • This invention provides peptides, compositions, and immunogenic compositions such as vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer, comprising administering immunogenic peptides.
  • the present invention provides an isolated WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:1-160, 162-185, 190, 191 and 193.
  • the present invention provides an isolated HLA class I binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183.
  • the present invention provides an isolated HLA class II binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:149, 156, 173, 174 and 180.
  • the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151
  • the HLA class II peptide consists of or comprises SEQ ID NO:149.
  • the present invention provides an isolated WT1 peptide having an amino acid (AA) sequence comprising any one of the sequences SEQ ID NO:1-53 or 43-XXX, or a fragment thereof.
  • the present invention provides an isolated HLA class I binding WT1 peptide having an amino acid (AA) sequence comprising of any one of the sequences SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183.
  • the present invention provides an isolated HLA class II binding WT1 peptide having an amino acid (AA) sequence comprising of any one of the sequences SEQ ID NO:149, 156, 173, 174 and 180.
  • the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151
  • the HLA class II peptide consists of or comprises SEQ ID NO:149.
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) a peptide selected from SEQ ID NO:1-160, 162-185, 190, 191 and 193.
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class I binding peptide selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183.
  • the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class II binding peptide selected from SEQ ID NO:149, 156, 173, 174 and 180.
  • the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151
  • the HLA class II peptide consists of or comprises SEQ ID NO:149.
  • the present invention provides a vaccine comprising one or more peptides of SEQ ID NO:1-160, 162-185, 190, 191 and 193.
  • the present invention provides a vaccine comprising one or more HLA class I binding peptides selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183.
  • the present invention provides a vaccine comprising one or more HLA class II binding peptides selected from SEQ ID NO:149, 156, 173, 174 and 180.
  • the present invention provides a vaccine comprising one or more HLA class I binding peptides selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, and one or more HLA class II binding peptides selected from SEQ ID NO:149, 156, 173, 174 and 180.
  • HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151
  • HLA class II peptide consists of or comprises SEQ ID NO:149.
  • the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.
  • the present invention provides a method of inducing an anti-cancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby inducing an anti-mesothelioma immune response in a subject.
  • the fragment of a WT1 protein consists of a peptide or comprises a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 and 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.
  • the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby treating a subject with a mesothelioma.
  • the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 and 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.
  • HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151
  • HLA class II peptide consists of or comprises SEQ ID NO:149.
  • the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject.
  • the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.
  • HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151
  • HLA class II peptide consists of or comprises SEQ ID NO:149.
  • the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.
  • the present invention provides a method of inducing an anti-cancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby inducing an anti-mesothelioma immune response in a subject.
  • the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193.
  • the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.
  • HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151
  • HLA class II peptide consists of or comprises SEQ ID NO:149.
  • the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby treating a subject with a mesothelioma.
  • the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193.
  • the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject.
  • the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193.
  • the cancer is a WT1-expressing cancer.
  • the WT1-expressing cancer is an acute myelogenous leukemia (AML).
  • the WT1-expressing cancer is associated with a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • the WT1-expressing cancer is an MDS.
  • the WT1-expressing cancer is a non-small cell lung cancer (NSCLC).
  • the WT1-expressing cancer is a Wilms' tumor.
  • the WT1-expressing cancer is a leukemia.
  • the WT1-expressing cancer is a hematological cancer.
  • the WT1-expressing cancer is a lymphoma.
  • the WT1-expressing cancer is a desmoplastic small round cell tumor.
  • the WT1-expressing cancer is a mesothelioma.
  • the WT1-expressing cancer is a malignant mesothelioma.
  • the WT1-expressing cancer is a gastric cancer.
  • the WT1-expressing cancer is a colon cancer.
  • the WT1-expressing cancer is a lung cancer.
  • the WT1-expressing cancer is abreast cancer.
  • the WT1-expressing cancer is a germ cell tumor.
  • the WT1-expressing cancer is an ovarian cancer.
  • the WT1-expressing cancer is a uterine cancer. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a liver cancer. In another embodiment, the WT1-expressing cancer is a renal cancer. In another embodiment, the WT1-expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT1-expressing cancer is a sarcoma. In another embodiment, the WT1-expressing cancer is any other carcinoma or sarcoma.
  • the WT1-expressing cancer is a solid tumor.
  • the solid tumor is associated with a WT1-expressing cancer.
  • the solid tumor is associated with a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • NSCLC non-small cell lung cancer
  • the solid tumor is associated with a lung cancer.
  • the solid tumor is associated with a breast cancer.
  • the solid tumor is associated with a colorectal cancer.
  • the solid tumor is associated with a prostate cancer.
  • the solid tumor is associated with an ovarian cancer.
  • the solid tumor is associated with a renal cancer.
  • the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
  • the present invention provides a composition comprising an isolated peptide of the invention in combination with at least 1 additional WT1 peptide.
  • a composition comprising at least 2 different isolated peptides of the present invention is provided.
  • a composition comprising at least 3 or at least 4 different isolated peptides of the present invention is provided.
  • Each possibility represents a separate embodiment of the present invention.
  • the composition of the present invention is a vaccine.
  • the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a peptide or composition of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a peptide or composition of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.
  • the present invention provides a method of inducing formation and proliferation of a WT1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of a WT1 protein-specific CTL.
  • the present invention provides a method of inducing formation and proliferation of (a) a WT1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT1 protein; or a combination thereof.
  • the invention is directed to a peptide of the invention with at least one amino acid change that increases the affinity of the peptide for binding to a HLA molecule.
  • “Peptide,” in another embodiment of methods and compositions of the present invention, refers to a compound of subunit AA connected by peptide bonds.
  • the peptide comprises an AA analogue.
  • the peptide comprises a peptidomimetic.
  • the different AA analogues and peptidomimetics that can be included in the peptides of methods and compositions of the present invention are enumerated herein below.
  • the subunits are, in another embodiment, linked by peptide bonds.
  • the subunit is linked by another type of bond, e.g. ester, ether, etc. Each possibility represents a separate embodiment of the present invention.
  • WT1 peptides The unaltered peptides of the present invention (as described both above and below) are referred to collectively herein as “WT1 peptides.” Each of the embodiments enumerated below for “WT1 peptides” applies to unaltered WT1 peptides and HLA class I and class II heteroclitic peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
  • a WT1 peptide of the present invention binds to an HLA class I molecule or a class II molecule. In another embodiment the peptide binds to both a class I and a class II molecule.
  • the HLA class II molecule is an HLA-DRB molecule.
  • the HLA class Il-molecule is an HLA-DRA molecule.
  • the HLA molecule is an HLA-DQA1 molecule.
  • the HLA molecule is an HLA-DQB1 molecule.
  • the HLA molecule is an HLA-DPA1 molecule.
  • the HLA molecule is an HLA-DPB 1 molecule.
  • the HLA molecule is an HLA-DMA molecule. In another embodiment, the HLA molecule is an HLA-DMB molecule. In another embodiment, the HLA molecule is an HLA-DOA molecule. In another embodiment, the HLA molecule is an HLA-DOB molecule. In another embodiment, the HLA molecule is any other HLA class Il-molecule known in the art. Each possibility represents a separate embodiment of the present invention.
  • the HLA class I molecule whose binding motif is contained in or comprising a peptide of the present invention is, in another embodiment, an HLA-A molecule.
  • the HLA class I molecule is an HLA-B molecule.
  • the HLA class I molecule is an HLA-C molecule.
  • the HLA class I molecule is an HLA-A0201 molecule.
  • the molecule is HLA A1.
  • the HLA class I molecule is HLA A2.
  • the HLA class I molecule is HLA A2.1.
  • the HLA class I molecule is HLA A3.
  • the HLA class I molecule is HLA A3.2.
  • the HLA class I molecule is HLA A11. In another embodiment, the HLA class I molecule is HLA A24. In another embodiment, the HLA class I molecule is HLA B7. In another embodiment, the HLA class I molecule is HLA B27. In another embodiment, the HLA class I molecule is HLA B8. Each possibility represents a separate embodiment of the present invention.
  • the HLA class I molecule-binding WT1 peptide of methods and compositions of the present invention binds to a superfamily of HLA class I molecules.
  • the superfamily is the A2 superfamily.
  • the superfamily is the A3 superfamily.
  • the superfamily is the A24 superfamily.
  • the superfamily is the B7 superfamily.
  • the superfamily is the B27 superfamily.
  • the superfamily is the B44 superfamily.
  • the superfamily is the C1 superfamily.
  • the superfamily is the C4 superfamily.
  • the superfamily is any other superfamily known in the art. Each possibility represents a separate embodiment of the present invention.
  • the HLA molecule is a A0101, A0201, A0203, A2402, A6901, B0702, A3101, B3501, B3503, B3508, B3802, B3801, B3901, B4001, B4402, B4701, B5701, C0401, C1701, DRB 1 0101, DRB 1 0402, DRB 1 0402, DRB 1 0401 or DRB 1 1104 molecule.
  • the peptides of SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, and SEQ ID NO:149, 156, 173, 174 and 180 bind to the HLA class I or class II molecules described for each peptide in Tables 1 or 2.
  • the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151
  • the HLA class II peptide consists of or comprises SEQ ID NO:149, and bind to the corresponding HLA molecule or molecules indicated for each peptide in Table 1 or Table 2.
  • certain peptides can bind to more than one HLA allele.
  • a modification of a peptide of the invention comprises at least one heteroclitic amino acid change, also referred to as a mutation or mutated, or an anchor residue mutation (see below).
  • An HLA class I molecule binding motif of a modified peptide of the present invention exhibits an increased affinity for the HLA class I molecule, relative to the unmutated counterpart of the peptide.
  • the point mutation increases the affinity of the isolated, mutated WT1 peptide for the HLA class I molecule.
  • the increase in affinity is relative to the affinity (for the same HLA class I molecule) of the isolated, unmutated WT1 peptide wherefrom the isolated, mutated WT1 peptide was derived.
  • Each possibility represents a separate embodiment of the present invention.
  • a WT1 peptide of methods and compositions of the present invention is so designed as to exhibit affinity for an HLA molecule.
  • the affinity is a high affinity, as described herein.
  • HLA molecules known in another embodiment as major histocompatibility complex (MHC) molecules, bind peptides and present them to immune cells.
  • MHC major histocompatibility complex
  • the immunogenicity of a peptide is partially determined by its affinity for HLA molecules.
  • HLA class I molecules interact with CD8 molecules, which are generally present on cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • HLA class II molecules interact with CD4 molecules, which are generally present on helper T lymphocytes.
  • a peptide of the present invention is immunogenic.
  • immunogenic refers to an ability to stimulate, elicit or participate in an immune response.
  • the immune response elicited is a cell-mediated immune response.
  • the immune response is a combination of cell-mediated and humoral responses.
  • T cells that bind to the MHC molecule-peptide complex become activated and induced to proliferate and lyse cells expressing a protein comprising the peptide.
  • T cells are typically initially activated by “professional” antigen presenting cells (“APC”; e.g. dendritic cells, monocytes, and macrophages), which present costimulatory molecules that encourage T cell activation as opposed to anergy or apoptosis.
  • APC antigen presenting cells
  • the response is heteroclitic, as described herein, such that the CTL lyses a neoplastic cell expressing a protein which has an AA sequence homologous to a peptide of this invention, or a different peptide than that used to first stimulate the T cell.
  • an encounter of a T cell with a peptide of this invention induces its differentiation into an effector and/or memory T cell. Subsequent encounters between the effector or memory T cell and the same peptide, or, in another embodiment, with a related peptide of this invention, leads to a faster and more intense immune response. Such responses are gauged, in another embodiment, by measuring the degree of proliferation of the T cell population exposed to the peptide. In another embodiment, such responses are gauged by any of the methods enumerated herein below.
  • the peptides of methods and compositions of the present invention bind an HLA class II molecule with high affinity.
  • the HLA class II molecule is any HLA class II molecule enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • derivatives of peptides of methods and compositions of the present invention bind an HLA class I molecule with high affinity.
  • the MHC class I molecule is any MHC class I molecule enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • a peptide of methods and compositions of the present invention binds an HLA class II molecule with significant affinity, while a peptide derived from the original peptide binds an HLA class I molecule with significant affinity.
  • affinity refers to the concentration of peptide necessary for inhibiting binding of a standard peptide to the indicated MHC molecule by 50%.
  • “high affinity” refers to an affinity is such that a concentration of about 500 nanomolar (nM) or less of the peptide is required for 50% inhibition of binding of a standard peptide. In another embodiment, a concentration of about 400 nM or less of the peptide is required.
  • the binding affinity is 300 nM. In another embodiment, the binding affinity is 200 nM. In another embodiment, the binding affinity is 150 nM. In another embodiment, the binding affinity is 100 nM. In another embodiment, the binding affinity is 80 nM. In another embodiment, the binding affinity is 60 nM.
  • the binding affinity is 40 nM. In another embodiment, the binding affinity is 30 nM. In another embodiment, the binding affinity is 20 nM. In another embodiment, the binding affinity is 15 nM. In another embodiment, the binding affinity is 10 nM. In another embodiment, the binding affinity is 8 nM. In another embodiment, the binding affinity is 6 nM. In another embodiment, the binding affinity is 4 nM. In another embodiment, the binding affinity is 3 nM. In another embodiment, the binding affinity is 2 nM. In another embodiment, the binding affinity is 1.5 nM. In another embodiment, the binding affinity is 1 nM. In another embodiment, the binding affinity is 0.8 nM. In another embodiment, the binding affinity is 0.6 nM. In another embodiment, the binding affinity is 0.5 nM. In another embodiment, the binding affinity is 0.4 nM. In another embodiment, the binding affinity is 0.3 nM. In another embodiment, the binding affinity is less than 0.3 nM.
  • affinity refers to a measure of binding strength to the MHC molecule.
  • affinity is measured using a method known in the art to measure competitive binding affinities.
  • affinity is measured using a method known in the art to measure relative binding affinities.
  • the method is a competitive binding assay.
  • the method is radioimmunoassay or RIA.
  • the method is BiaCore analyses.
  • the method is any other method known in the art.
  • the method yields an IC50 in relation to an IC50 of a reference peptide of known affinity.
  • “high affinity” refers to an IC50 of 0.5-500 nM.
  • the IC50 is 1-300 nM. In another embodiment, the IC50 is 1.5-200 nM. In another embodiment, the IC50 is 2-100 nM. In another embodiment, the IC50 is 3-100 nM. In another embodiment, the IC50 is 4-100 nM. In another embodiment, the IC50 is 6-100 nM. In another embodiment, the IC50 is 10-100 nM. In another embodiment, the IC50 is 30-100 nM. In another embodiment, the IC50 is 3-80 nM. In another embodiment, the IC50 is 4-60 nM. In another embodiment, the IC50 is 5-50 nM.
  • the IC50 is 6-50 nM. In another embodiment, the IC50 is 8-50 nM. In another embodiment, the IC50 is 10-50 nM. In another embodiment, the IC50 is 20-50 nM. In another embodiment, the IC50 is 6-40 nM. In another embodiment, the IC50 is 8-30 nM. In another embodiment, the IC50 is 10-25 nM. In another embodiment, the IC50 is 15-25 nM.
  • Each affinity and range of affinities represents a separate embodiment of the present invention.
  • a peptide of methods and compositions of the present invention binds to a superfamily of HLA molecules.
  • Superfamilies of HLA molecules share very similar or identical binding motifs.
  • the superfamily is a HLA class I superfamily.
  • the superfamily is a HLA class II superfamily. Each possibility represents a separate embodiment of the present invention.
  • HLA-binding peptide refers, in another embodiment, to a peptide that binds an HLA molecule with measurable affinity. In another embodiment, the terms refer to a peptide that binds an HLA molecule with high affinity. In another embodiment, the terms refer to a peptide that binds an HLA molecule with sufficient affinity to activate a T cell precursor. In another embodiment, the terms refer to a peptide that binds an HLA molecule with sufficient affinity to mediate recognition by a T cell.
  • the HLA molecule is, in other embodiments, any of the HLA molecules enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • Heteroclitic refers, in another embodiment, to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived (e.g. the peptide not containing the anchor residue mutations).
  • original peptide refers to a peptide of the present invention.
  • heteroclitic refers to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide.
  • a “heteroclitic” immune response refers to an immune response that recognizes the original peptide from which the improved peptide was derived (e.g. the peptide not containing the anchor residue mutations).
  • a “heteroclitic” immune response refers to an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide.
  • the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response substantially equal to the response to vaccination with the original peptide.
  • a heteroclitic peptide of the present invention is an HLA class I heteroclitic peptide.
  • a heteroclitic peptide of the present invention induces an immune response that is increased at least 2-fold relative to the WT1 peptide from which the heteroclitic peptide was derived (“native peptide”).
  • the increase is 3-fold relative to the native peptide.
  • the increase is 5-fold relative to the native peptide.
  • the increase is 7-fold relative to the native peptide.
  • the increase is 10-fold relative to the native peptide.
  • the increase is 15-fold relative to the native peptide.
  • the increase is 20-fold relative to the native peptide.
  • the increase is 30-fold relative to the native peptide.
  • the increase is 50-fold relative to the native peptide. In another embodiment, the increase is 100-fold relative to the native peptide. In another embodiment, the increase is 150-fold relative to the native peptide. In another embodiment, the increase is 200-fold relative to the native peptide. In another embodiment, the increase is 300-fold relative to the native peptide. In another embodiment, the increase is 500-fold relative to the native peptide. In another embodiment, the increase is 1000-fold relative to the native peptide. In another embodiment, the increase is more than 1000-fold relative to the native peptide. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides a HLA class II heteroclitic peptide derived from an isolated WT1 peptide of the present invention.
  • the process of deriving comprises introducing a mutation that enhances a binding of the peptide to an HLA class II molecule.
  • the process of deriving consists of introducing a mutation that enhances a binding of the peptide to an HLA class I molecule.
  • the mutation is in an HLA class II anchor residue.
  • a heteroclitic class II peptide of the present invention is identified and tested in a manner analogous to identification and testing of HLA class I heteroclitic peptides, as exemplified herein. Each possibility represents a separate embodiment of the present invention.
  • the HLA class II binding site in a peptide of the present invention is created or improved by mutation of an HLA class II motif anchor residue.
  • the anchor residue that is modified is in the P1 position.
  • the anchor residue is at the P2 position.
  • the anchor residue is at the P6 position.
  • the anchor residue is at the P9 position.
  • the anchor residue is selected from the P1, P2, P6, and P9 positions.
  • the anchor residue is at the P3 position.
  • the anchor residue is at the P4 position.
  • the anchor residue is at the P5 position.
  • the anchor residue is at the P6 position.
  • the anchor residue is at the P8 position.
  • the anchor residue is at the P10 position. In another embodiment, the anchor residue is at the P11 position. In another embodiment, the anchor residue is at the P12 position. In another embodiment, the anchor residue is at the P13 position. In another embodiment, the anchor residue is at any other anchor residue of an HLA class II molecule that is known in the art. In another embodiment, residues other than P1, P2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve HLA class II binding. Each possibility represents a separate embodiment of the present invention.
  • a heteroclitic peptide is generated by introduction of a mutation that creates an anchor motif.
  • Anchor motifs or “anchor residues” refers, in another embodiment, to 1 or a set of preferred residues at particular positions in an HLA-binding sequence.
  • HLA-binding sequence is an HLA class II-binding sequence.
  • HLA-binding sequence is an HLA class I-binding sequence.
  • the positions corresponding to the anchor motifs are those that play a significant role in binding the HLA molecule.
  • the anchor residue is a primary anchor motif.
  • the anchor residue is a secondary anchor motif.
  • MHC class II epitope is predicted using TEPITOPE (Meister G E, Roberts C G et al, Vaccine 1995 13: 581-91).
  • MHC class II epitope is predicted using EpiMatrix (De Groot A S, Jesdale B M et al, AIDS Res Hum Retroviruses 1997 13: 529-31).
  • the MHC class II epitope is predicted using the Predict Method (Yu K, Petrovsky N et al, Mol Med. 2002 8: 137-48).
  • the MHC class II epitope is predicted using the SYFPEITHI epitope prediction algorithm (Examples). In another embodiment, the MHC class II epitope is predicted using Rankpep. In another embodiment, the MHC class II epitope is predicted using any other method known in the art. Each possibility represents a separate embodiment of the present invention.
  • the anchor residue that is modified is in the P1 position.
  • the anchor residue is in the P2 position.
  • the anchor residue is in the P6 position.
  • the anchor residue is in the P9 position.
  • the anchor residue is the P3, P4, P5, P6, P8, P10, P11, P12, or P13 position.
  • the anchor residue is any other anchor residue of an HLA class II molecule that is known in the art.
  • residues other than P1, P2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve HLA class II binding.
  • any combination of the above residues is mutated.
  • Each possibility represents a separate embodiment of the present invention.
  • a WT1 peptide of the present invention binds to 2 distinct HLA class II molecules. In another embodiment, the peptide binds to three distinct HLA class II molecules. In another embodiment, the peptide binds to four distinct HLA class II molecules. In another embodiment, the peptide binds to five distinct HLA class II molecules. In another embodiment, the peptide binds to six distinct HLA class II molecules. In another embodiment, the peptide binds to more than six distinct HLA class II molecules.
  • the HLA class II molecules that are bound by a WT1 peptide of the present invention are encoded by two or more distinct alleles at a given HLA class II locus. In another embodiment, the HLA class II molecules are encoded by 3 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 4 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 5 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 6 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by more than six distinct alleles at a locus.
  • the HLA class II molecules bound by the WT1 peptide are encoded by HLA class II genes at 2 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 2 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 3 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 3 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 4 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 4 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at more than 4 distinct loci.
  • the loci are selected from HLA-DRB loci.
  • the HLA class II-binding peptide is an HLA-DRA binding peptide.
  • the peptide is an HLA-DQA1 binding peptide.
  • the peptide is an HLA-DQB 1 binding peptide.
  • the peptide is an HLA-DPA1 binding peptide.
  • the peptide is an HLA-DPB 1 binding peptide.
  • the peptide is an HLA-DMA binding peptide.
  • the peptide is an HLA-DMB binding peptide.
  • the peptide is an HLA-DOA binding peptide.
  • the peptide is an HLA-DOB binding peptide.
  • the peptide binds to any other HLA class II molecule known in the art. Each possibility represents a separate embodiment of the present invention.
  • a WT1 peptide of the present invention binds to 2 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 3 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 4 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 5 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 6 distinct HLA-DRB molecules. In another embodiment, the peptide binds to more than 6 distinct HLA-DRB molecules.
  • a WT1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by 3 distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by 4 distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by 5 distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by 6 distinct HLA-DRB alleles.
  • the HLA-DRB molecules are encoded by more than 6 distinct HLA-DRB alleles. Each possibility represents a separate embodiment of the present invention.
  • a WT1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501.
  • the WT1 peptide binds to HLA-DRB molecules encoded by 3 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501.
  • the WT1 peptide binds to HLA-DRB molecules encoded by 4 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by 5 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, DRB 1104 and DRB 1501.
  • the WT1 peptide binds to HLA-DRB molecules encoded by each of the following HLA-DRB alleles: DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501.
  • HLA-DRB alleles DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501.
  • the present invention provides a composition comprising 2 distinct WT1 peptides of the present invention.
  • the 2 distinct WT1 peptides are both unaltered.
  • 1 of the WT1 peptides is unaltered, while the other is heteroclitic.
  • both of the WT1 peptides are heteroclitic.
  • the composition comprises 3 distinct WT1 peptides of the present invention. In another embodiment, the composition comprises 4 distinct WT1 peptides of the present invention. In another embodiment, the composition comprises 5 distinct WT1 peptides of the present invention. In another embodiment, the composition comprises more than 5 distinct isolated WT1 peptides of the present invention.
  • 2 of the WT1 peptides in the composition are unaltered. In another embodiment, 2 of the WT1 peptides in the composition are heteroclitic. In another embodiment, 2 of the WT1 peptides in the composition are unaltered, and 2 are heteroclitic. In another embodiment, more than 2 of the WT1 peptides in the composition are unaltered. In another embodiment, more than 2 of the WT1 peptides in the composition are heteroclitic. In another embodiment, more than 2 of the WT1 peptides in the composition are unaltered, and more than 2 are heteroclitic. Each possibility represents a separate embodiment of the present invention.
  • 1 of the additional WT1 peptides in a composition of the present invention has a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193.
  • 2 of the additional WT1 peptides have a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193.
  • 3 of the additional WT1 peptides have a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193.
  • any other immunogenic WT1 peptide known in the art is utilized as an additional WT1 peptide.
  • any combination of immunogenic WT1 peptides known in the art is utilized.
  • Non-limiting sources of other WT peptides include WO2005053618, WO2007047764 and WO2007120673.
  • Each additional WT1 peptide, and each combination thereof, represents a separate embodiment of the present invention.
  • a composition of the present invention contains 2 HLA class II heteroclitic peptides that are derived from the same isolated WT1 peptide of the present invention.
  • the 2 HLA class II heteroclitic peptides contain mutations in different HLA class II molecule anchor residues.
  • the 2 HLA class II heteroclitic peptides contain different mutations in the same anchor residues.
  • 2 of the HLA class II heteroclitic peptides are derived from different isolated WT1 peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
  • 2 WT1 peptides of the present invention, or the WT1 peptides that correspond to two HLA class II heteroclitic peptides of the present invention overlap with one another.
  • the overlap between the peptides is at least 7 amino acids (AA).
  • the overlap is at least 8 AA.
  • the overlap is at least 9 AA.
  • the overlap is 7 AA.
  • the overlap is 8 AA.
  • the overlap is 9 AA.
  • the overlap is 10 AA.
  • the overlap is 11 AA.
  • the overlap is 12 AA.
  • the overlap is 13 AA.
  • the overlap is 14 AA.
  • the overlap is 15 AA.
  • the overlap is 16 AA.
  • the overlap is more than 16 AA.
  • the peptides in a composition of the present invention bind to 2 distinct HLA class II molecules. In another embodiment, the peptides bind to 3 distinct HLA class II molecules. In another embodiment, the peptides bind to 4 distinct HLA class II molecules. In another embodiment, the peptides bind to 5 distinct HLA class II molecules. In another embodiment, the peptides bind to more than 5 distinct HLA class II molecules. In another embodiment, the peptides in the composition bind to the same HLA class II molecules.
  • each of the WT 1 peptides in a composition of the present invention binds to a set of HLA class II molecules. In another embodiment, each of the WT1 peptides binds to a distinct set of HLA class II molecules. In another embodiment, the WT1 peptides in the composition bind to the same set of HLA class II molecules. In another embodiment, 2 of the WT1 peptides bind to a distinct but overlapping set of HLA class II molecules. In another embodiment, 2 or more of the WT1 peptides bind to the same set of HLA class II molecules, while another of the WT1 peptides binds to a distinct set. In another embodiment, 2 or more of the WT1 peptides bind to an overlapping set of HLA class II molecules, while another of the WT1 peptides binds to a distinct set.
  • 2 or more of the WT1 peptides in a composition of the present invention each binds to more than 1 HLA-DRB molecule.
  • the 4 or more HLA-DRB molecules bound by the peptides in the composition are distinct from one another.
  • the HLA-DRB molecules are encoded by different HLA-DRB alleles. Each possibility represents a separate embodiment of the present invention.
  • HLA-DRB molecules 2 or more of the HLA class II molecules bound by WT1 peptides in a composition of the present invention are HLA-DRB molecules. In another embodiment, 3 or more of the HLA class II molecules that are bound are HLA-DRB molecules. In other embodiments, the HLA class II molecules that are bound can be any of the HLA class II molecules enumerated herein. In another embodiment, the HLA class II molecules that are bound are encoded by 2 or more distinct HLA class II alleles at a given locus. In another embodiment, the HLA class II molecules that are bound are encoded by HLA class II genes at 2 or more distinct loci.
  • compositions represents a separate embodiment of the present invention.
  • a “set of HLA class II molecules” refers to the HLA class II molecules encoded by different alleles at a particular locus.
  • the term refers to HLA class II molecules with a particular binding specificity.
  • the term refers to HLA class II molecules with a particular peptide consensus sequence.
  • the term refers to a superfamily of HLA class II molecules. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides a composition comprising an unaltered HLA class II molecule-binding WT1 peptide of the present invention and a second, HLA class I molecule-binding WT1 peptide.
  • the composition comprises more than 1 HLA class II molecule-binding WT1 peptide of the present invention, in addition to the HLA class I molecule-binding WT1 peptide.
  • the composition comprises more than 1 HLA class I molecule-binding WT1 peptide, in addition to the HLA class II molecule-binding WT1 peptide.
  • the AA sequence of the HLA class I molecule-binding WT1 peptide comprises a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193.
  • the AA sequence of the HLA class I molecule-binding WT1 peptide is selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193. Each possibility represents a separate embodiment of the present invention.
  • the HLA class I molecule-binding WT1 peptide is an HLA class I heteroclitic peptide.
  • the HLA class I molecule-binding WT1 peptide contains a mutation in an HLA class I molecule anchor residue thereof, as described further herein.
  • WT1-derived peptides were modified in HLA anchor residues to generate heteroclitic peptides with increased predicted binding to HLA-A0201 and HLA-A0301. Peptides with increased predicted binding also exhibited enhanced ability to bind HLA class I molecules and increased immunogenicity.
  • the mutation that enhances MHC binding is in the residue at position 1 of the HLA class I heteroclitic peptide.
  • the residue is changed to tyrosine.
  • the residue is changed to glycine.
  • the residue is changed to threonine.
  • the residue is changed to phenylalanine.
  • the residue is changed to any other residue known in the art.
  • a substitution in position 1 e.g. to tyrosine
  • the mutation is in position 2 of the HLA class I heteroclitic peptide.
  • the residue is changed to leucine.
  • the residue is changed to valine.
  • the residue is changed to isoleucine.
  • the residue is changed to methionine.
  • the residue is changed to any other residue known in the art.
  • the mutation is in position 6 of the HLA class I heteroclitic peptide.
  • the residue is changed to valine.
  • the residue is changed to cysteine.
  • the residue is changed to glutamine.
  • the residue is changed to histidine.
  • the residue is changed to any other residue known in the art.
  • the mutation is in position 9 of the HLA class I heteroclitic peptide.
  • the mutation changes the residue at the C-terminal position thereof.
  • the residue is changed to valine.
  • the residue is changed to threonine.
  • the residue is changed to isoleucine.
  • the residue is changed to leucine.
  • the residue is changed to alanine.
  • the residue is changed to cysteine.
  • the residue is changed to any other residue known in the art.
  • the point mutation is in a primary anchor residue.
  • the HLA class I primary anchor residues are positions 2 and 9.
  • the point mutation is in a secondary anchor residue.
  • the HLA class I secondary anchor residues are positions 1 and 8.
  • the HLA class I secondary anchor residues are positions 1, 3, 6, 7, and 8.
  • the point mutation is in a position selected from positions 4, 5, and 8. Each possibility represents a separate embodiment of the present invention.
  • the point mutation is in 1 or more residues in positions selected from positions 1, 2, 8, and 9 of the HLA class I binding motif. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 3, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 2, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 2, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 3, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions selected from positions 2 and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 6 and 9. Each possibility represents a separate embodiment of the present invention.
  • the HLA class I molecule-binding WT peptide has length of 9 AA. In another embodiment, the peptide has length of 10 AA. As provided herein, native and heteroclitic peptides of 9-10 AA exhibited substantial binding to HLA class I molecules and ability to elicit cytokine secretion and cytolysis by CTL.
  • the HLA class I molecule that is bound by the HLA class I molecule-binding WT1 peptide is an HLA-A molecule.
  • the HLA class I-molecule is an HLA-A2 molecule.
  • the HLA class I-molecule is an HLA-A3 molecule.
  • the HLA class I-molecule is an HLA-A11 molecule.
  • the HLA class I-molecule is an HLA-B 8 molecule.
  • the HLA class I-molecule is an HLA-0201 molecule.
  • the HLA class I-molecule binds any other HLA class I molecule known in the art. Each possibility represents a separate embodiment of the present invention.
  • a WT1 peptide of methods and compositions of the present invention has a length of 8-30 amino acids.
  • the peptide has a length of 9-11 AA.
  • the peptide ranges in size from 7-25 AA, or in another embodiment, 8-11, or in another embodiment, 8-15, or in another embodiment, 9-20, or in another embodiment, 9-18, or in another embodiment, 9-15, or in another embodiment, 8-12, or in another embodiment, 9-11 AA in length.
  • the peptide is 8 AA in length, or in another embodiment, 9 AA or in another embodiment, 10 AA or in another embodiment, 12 AA or in another embodiment, 25 AA in length, or in another embodiment, any length therebetween.
  • the peptide is of greater length, for example 50, or 100, or more.
  • the cell processes the peptide to a length of 7 and 25 AA in length.
  • the cell processes the peptide to a length of 9-11 AA Each possibility represents a separate embodiment of the present invention.
  • the peptide is 15-23 AA in length. In another embodiment, the length is 15-24 AA. In another embodiment, the length is 15-25 AA. In another embodiment, the length is 15-26 AA. In another embodiment, the length is 15-27 AA. In another embodiment, the length is 15-28 AA. In another embodiment, the length is 14-30 AA. In another embodiment, the length is 14-29 AA. In another embodiment, the length is 14-28 AA. In another embodiment, the length is 14-26 AA. In another embodiment, the length is 14-24 AA. In another embodiment, the length is 14-22 AA. In another embodiment, the length is 14-20 AA. In another embodiment, the length is 16-30 AA. In another embodiment, the length is 16-28 AA.
  • the length is 16-26 AA. In another embodiment, the length is 16-24 AA. In another embodiment, the length is 16-22 AA. In another embodiment, the length is 18-30 AA. In another embodiment, the length is 18-28 AA. In another embodiment, the length is 18-26 AA. In another embodiment, the length is 18-24 AA. In another embodiment, the length is 18-22 AA. In another embodiment, the length is 18-20 AA. In another embodiment, the length is 20-30 AA. In another embodiment, the length is 20-28 AA. In another embodiment, the length is 20-26 AA. In another embodiment, the length is 20-24 AA. In another embodiment, the length is 22-30 AA. In another embodiment, the length is 22-28 AA. In another embodiment, the length is 22-26 AA. In another embodiment, the length is 24-30 AA. In another embodiment, the length is 24-28 AA. In another embodiment, the length is 24-26 AA. In another embodiment, the length is 24-26 AA. In another embodiment, the length is 24-26 AA
  • minor modifications are made to peptides of the present invention without decreasing their affinity for HLA molecules or changing their TCR specificity, utilizing principles well known in the art.
  • “minor modifications” refers, in another embodiment, to e.g. insertion, deletion, or substitution of one AA, inclusive, or deletion or addition of 1-3 AA outside of the residues between 2 and 9, inclusive.
  • the computer algorithms described herein are useful for predicting the MHC class I-binding potential of peptides, they have 60-80% predictive accuracy; and thus, the peptides should be evaluated empirically before a final determination of MHC class I-binding affinity is made.
  • peptides of the present invention are not limited to peptides predicated by the algorithms to exhibit strong MHC class I-binding affinity.
  • the types are modifications that can be made are listed below. Each modification represents a separate embodiment of the present invention.
  • a peptide enumerated in the Examples of the present invention is further modified by mutating an anchor residue to an MHC class I preferred anchor residue, which can be, in other embodiments, any of the anchor residues enumerated herein.
  • a peptide of the present invention containing an MHC class I preferred anchor residue is further modified by mutating the anchor residue to a different MHC class I preferred residue for that location.
  • the different preferred residue can be, in other embodiments, any of the preferred residues enumerated herein.
  • the anchor residue that is further modified is in the 1 position. In another embodiment, the anchor residue is in the 2 position. In another embodiment, the anchor residue is in the 3 position. In another embodiment, the anchor residue is in the 4 position. In another embodiment, the anchor residue is in the 5 position. In another embodiment, the anchor residue is in the 6 position. In another embodiment, the anchor residue is in the 7 position. In another embodiment, the anchor residue is in the 8 position. In another embodiment, the anchor residue is in the 9 position. In the case of HLA class I-binding peptides, residues other than 2 and 9 can serve as secondary anchor residues; therefore, mutating them can improve MHC class I binding. Each possibility represents a separate embodiment of the present invention.
  • a peptide of methods and compositions of the present invention is a length variant of a peptide enumerated in the Examples.
  • the length variant is one amino acid (AA) shorter than the peptide from the Examples.
  • the length variant is two AA shorter than the peptide from the Examples.
  • the length variant is more than two AA shorter than the peptide from the Examples.
  • the shorter peptide is truncated on the N-terminal end.
  • the shorter peptide is truncated on the C-terminal end.
  • the truncated peptide is truncated on both the N-terminal and C-terminal ends.
  • Peptides are, in another embodiment, amenable to truncation without changing affinity for HLA molecules, as is well known in the art.
  • the length variant is longer than a peptide enumerated in the Examples of the present invention.
  • the longer peptide is extended on the N-terminal end in accordance with the surrounding WT1 sequence.
  • Peptides are, in another embodiment, amenable to extension on the N-terminal end without changing affinity for HLA molecules, as is well known in the art. Such peptides are thus equivalents of the peptides enumerated in the Examples.
  • the N-terminal extended peptide is extended by one residue.
  • the N-terminal extended peptide is extended by two residues.
  • the N-terminal extended peptide is extended by three residues.
  • the N-terminal extended peptide is extended by more than three residues.
  • the longer peptide is extended on the C terminal end in accordance with the surrounding WT1 sequence.
  • Peptides are, in another embodiment, amenable to extension on the C-terminal end without changing affinity for HLA molecules, as is well known in the art. Such peptides are thus equivalents of the peptides enumerated in the Examples of the present invention.
  • the C-terminal extended peptide is extended by one residue.
  • the C-terminal extended peptide is extended by two residues.
  • the C-terminal extended peptide is extended by three residues.
  • the C-terminal extended peptide is extended by more than three residues.
  • the extended peptide is extended on both the N-terminal and C-terminal ends in accordance with the surrounding WT1 sequence.
  • a truncated peptide of the present invention retains the HLA anchor residues (e.g. the HLA class I anchor residues) on the second residue and the C-terminal residue, with a smaller number of intervening residues (e.g. 5) than a peptide enumerated in the Examples of the present invention.
  • Peptides are, in another embodiment, amenable to such mutation without changing affinity for
  • HLA molecules In another embodiment, such a truncated peptide is designed by removing one of the intervening residues of one of the above sequences.
  • the HLA anchor residues are retained on the second and eighth residues.
  • the HLA anchor residues are retained on the first and eighth residues.
  • an extended peptide of the present invention retains the HLA anchor residues (e.g. the HLA class I anchor residues) on the second residue and the C-terminal residue, with a larger number of intervening residues (e.g. 7 or 8) than a peptide enumerated in the Examples of the present invention.
  • such an extended peptide is designed by adding one or more residues between two of the intervening residues of one of the above sequences. It is well known in the art that residues can be removed from or added between the intervening sequences of HLA-binding peptides without changing affinity for HLA. Such peptides are thus equivalents of the peptides enumerated in the Examples of the present invention.
  • the HLA anchor residues are retained on the second and ninth residues. In another embodiment, the HLA anchor residues are retained on the first and eighth residues. In another embodiment, the HLA anchor residues are retained on the two residues separated by six intervening residues. Each possibility represents a separate embodiment of the present invention.
  • “Fragment,” in another embodiment, refers to a peptide of 11 or more AA in length.
  • a peptide fragment of the present invention is 16 or more AA long.
  • the fragment is 12 or more AA long.
  • the fragment is 13 or more AA.
  • the fragment is 14 or more AA.
  • the fragment is 15 or more AA.
  • the fragment is 17 or more AA.
  • the fragment is 18 or more AA.
  • the fragment is 19 or more AA.
  • the fragment is 22 or more AA.
  • the fragment is 8-12 AA.
  • the fragment is about 8-12 AA.
  • the fragment is 16-19 AA.
  • the fragment is about 16-19 AA.
  • the fragment is about 16-19 AA.
  • the fragment is about 16-19 AA.
  • the fragment is about 16-19 AA.
  • the fragment is about 16-19 AA.
  • the fragment is about 16-19 AA.
  • the fragment is about 16-19 AA.
  • the fragment is about 16-19 AA.
  • “Fragment of a WT1 protein,” in another embodiment, refers to any of the definitions of “fragment” found herein. Each definition represents a separate embodiment of the present invention.
  • a peptide of the present invention is homologous to a peptide enumerated in the Examples.
  • Homology is, in another embodiment, determined by computer algorithm for sequence alignment, by methods well described in the art.
  • computer algorithm analysis of nucleic acid sequence homology includes the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.
  • “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 70%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 75%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 78%.
  • “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 82%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 85%.
  • “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 87%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than [0128] 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 92%.
  • “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 95%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 97%.
  • homology refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of 100%. Each possibility represents a separate embodiment of the present invention. [00114] In another embodiment, homology is determined via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, “Nucleic Acid Hybridization” Hames, B. D., and Higgins S.
  • methods of hybridization are carried out under moderate to stringent conditions, to the complement of a DNA encoding a native caspase peptide.
  • Hybridization conditions being, for example, overnight incubation at 42° C.
  • the present invention provides a composition comprising a peptide of this invention.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the composition further comprises an adjuvant.
  • the composition comprises 2 or more peptides of the present invention.
  • the composition further comprises any of the additives, compounds, or excipients set forth hereinbelow.
  • the adjuvant is KLH, QS21, Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG or alum.
  • the carrier is any carrier enumerated herein.
  • the adjuvant is any adjuvant enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • this invention provides a vaccine comprising a peptide of this invention.
  • this invention provides a vaccine comprising an antigen-presenting cell (APC) and a peptide of this invention.
  • the vaccine further comprises a carrier.
  • the vaccine further comprises an adjuvant.
  • the vaccine further comprises an APC.
  • the vaccine further comprises a combination of more than 1 of an antigen, carrier, and/or APC.
  • the vaccine is a cell-based composition. Each possibility represents a separate embodiment of the present invention.
  • the term “vaccine” refers to a material or composition that, when introduced into a subject, provides a prophylactic or therapeutic response for a particular disease, condition, or symptom of same.
  • this invention comprises peptide-based vaccines, wherein the peptide comprises any embodiment listed herein, including immunomodulating compounds such as cytokines, adjuvants, etc.
  • a vaccine of methods and compositions of the present invention further comprises an adjuvant.
  • the adjuvant is a Montanide.
  • the adjuvant is Montanide ISA 51.
  • Montanide ISA 51 contains a natural metabolizable oil and a refined emulsifier.
  • the adjuvant is GM-CSF.
  • Recombinant GM-CSF is a human protein grown, in another embodiment, in a yeast ( S. cerevisiae ) vector.
  • GM-CSF promotes clonal expansion and differentiation of hematopoietic progenitor cells, APC, and dendritic cells and T cells.
  • the adjuvant is a cytokine. In another embodiment, the adjuvant is a growth factor. In another embodiment, the adjuvant is a cell population. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is Freund's incomplete adjuvant. In another embodiment, the adjuvant is aluminum phosphate. In another embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is BCG. In another embodiment, the adjuvant is alum.
  • the adjuvant is an interleukin. In another embodiment, the adjuvant is a chemokine. In another embodiment, the adjuvant is any other type of adjuvant known in the art.
  • the WT1 vaccine comprises two the above adjuvants. In another embodiment, the WT1 vaccine comprises more than two the above adjuvants. Each possibility represents a separate embodiment of the present invention.
  • a vaccine or composition of the present invention can comprise any of the embodiments of WT1 peptides of the present invention and combinations thereof. Each possibility represents a separate embodiment of the present invention.
  • the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • the present invention provides a method of treating a subject with an MDS, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby treating a subject with an MDS.
  • the present invention provides a method of suppressing or halting the progression of a WT1-expressing cancer in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby suppressing or halting the progression of a WT1-expressing cancer.
  • the present invention provides a method of reducing the incidence of a WT1-expressing cancer in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of a WT1-expressing cancer in a subject.
  • the present invention provides a method of reducing the incidence of an AML in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of an AML.
  • the present invention provides a method of reducing the incidence of relapse of a WT1-expressing cancer in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of relapse of a WT1-expressing cancer in a subject.
  • the present invention provides a method of reducing the incidence of relapse of an AML in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of relapse of an AML in a subject.
  • the present invention provides a method of breaking a T cell tolerance of a subject to a WT1-expressing cancer, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby breaking a T cell tolerance to a WT1-expressing cancer.
  • the present invention provides a method of treating a subject having a WT1-expressing cancer, comprising (a) inducing in a donor formation and proliferation of human cytotoxic T lymphocytes (CTL) that recognize a malignant cell of the cancer by a method of the present invention; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer.
  • CTL cytotoxic T lymphocytes
  • the present invention provides a method of treating a subject having a WT 1-expressing cancer, comprising (a) inducing ex vivo formation and proliferation of human CTL that recognize a malignant cell of the cancer by a method of the present invention, wherein the human immune cells are obtained from a donor; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer.
  • the present invention provides a method of inducing formation and proliferation of (a) a WT1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT1 protein-specific CD8 + lymphocyte; or (b) a CD4 + lymphocyte specific for the WT1 protein; or a combination thereof.
  • This method can be conducted in vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect.
  • the present invention provides a method of inducing the formation and proliferation of CTL specific for cells of a WT1-expressing cancer, the method comprising contacting a lymphocyte population with a vaccine of the present invention.
  • the vaccine is an APC associated with a peptide of the present invention.
  • the vaccine is an APC associated with a mixture of peptides of the present invention.
  • this invention provides a method of generating a heteroclitic immune response in a subject, wherein the heteroclitic immune response is directed against a WT1-expressing cancer, the method comprising administering to the subject a vaccine of the present invention, thereby generating a heteroclitic immune response.
  • the present invention provides a method of inducing an anti-mesothelioma immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT1 protein; or (b) a fragment of a WT protein, thereby inducing an anti-mesothelioma immune response in a subject.
  • an immunogenic composition comprising (a) a WT1 protein; or (b) a fragment of a WT protein, thereby inducing an anti-mesothelioma immune response in a subject.
  • the mesothelioma is a malignant mesothelioma.
  • the present invention provides a method of inducing an anti-mesothelioma immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising a nucleotide molecule encoding (a) a WT1 protein; or (b) a fragment of a WT1 protein, thereby inducing an anti-mesothelioma immune response in a subject.
  • the mesothelioma is a malignant mesothelioma.
  • the present invention provides a method of treating a subject with a mesothelioma, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; or (b) a fragment of a WT protein, thereby treating a subject with a mesothelioma.
  • the mesothelioma is a malignant mesothelioma.
  • the present invention provides a method of treating a subject with a mesothelioma, the method comprising the step of administering to the subject an immunogenic composition comprising a nucleotide molecule encoding (a) a WT1 protein; or (b) a fragment of a WT1 protein, thereby treating a subject with a mesothelioma.
  • the mesothelioma is a malignant mesothelioma.
  • the present invention provides a method of reducing an incidence of a mesothelioma, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; or (b) a fragment of a WT protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject.
  • the mesothelioma is a malignant mesothelioma.
  • the present invention provides a method of reducing an incidence of a mesothelioma, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising a nucleotide molecule encoding (a) a WT1 protein; or (b) a fragment of a WT1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject.
  • the mesothelioma is a malignant mesothelioma.
  • a target cell of an immune response elicited by a method of the present invention presents the WT1 peptide of the present invention, or a corresponding WT1 fragment, on an HLA molecule.
  • the HLA molecule is an HLA class I molecule.
  • the HLA molecule is any HLA class I subtype or HLA class I molecule known in the art.
  • the immune response against the WT1 peptide or fragment is a heteroclitic immune response. Each possibility represents a separate embodiment of the present invention.
  • the WT1-expressing cancer is an acute myelogenous leukemia (AML). In another embodiment, the WT1-expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT1-expressing cancer is an MDS. In another embodiment, the WT1-expressing cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the WT1-expressing cancer is a Wilms' tumor. In another embodiment, the WT1-expressing cancer is a leukemia. In another embodiment, the WT1-expressing cancer is a hematological cancer. In another embodiment, the WT1-expressing cancer is a lymphoma.
  • AML acute myelogenous leukemia
  • MDS myelodysplastic syndrome
  • the WT1-expressing cancer is an MDS.
  • the WT1-expressing cancer is a non-small cell lung cancer (NSCLC).
  • the WT1-expressing cancer is a Wilms' tumor.
  • the WT1-expressing cancer is a desmoplastic small round cell tumor.
  • the WT1-expressing cancer is a mesothelioma.
  • the WT1-expressing cancer is a malignant mesothelioma.
  • the WT1-expressing cancer is a gastric cancer.
  • the WT1-expressing cancer is a colon cancer.
  • the WT1-expressing cancer is a lung cancer.
  • the WT1-expressing cancer is abreast cancer.
  • the WT1-expressing cancer is a germ cell tumor.
  • the WT1-expressing cancer is an ovarian cancer.
  • the WT 1-expressing cancer is a uterine cancer. In another embodiment, the WT 1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a liver cancer. In another embodiment, the WT1-expressing cancer is a renal cancer. In another embodiment, the WT1-expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT1-expressing cancer is a sarcoma. In another embodiment, the WT1-expressing cancer is any other carcinoma or sarcoma.
  • the WT1-expressing cancer is a solid tumor.
  • the solid tumor is associated with a WT1-expressing cancer.
  • the solid tumor is associated with a myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • NSCLC non-small cell lung cancer
  • the solid tumor is associated with a lung cancer.
  • the solid tumor is associated with a breast cancer.
  • the solid tumor is associated with a colorectal cancer.
  • the solid tumor is associated with a prostate cancer.
  • the solid tumor is associated with an ovarian cancer.
  • the solid tumor is associated with a renal cancer.
  • the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
  • a cancer or tumor treated by a method of the present invention is suspected to express WT1.
  • WT1 expression has not been verified by testing of the actual tumor sample.
  • the cancer or tumor is of a type known to express WT1 in many cases. In another embodiment, the type expresses WT1 in the majority of cases.
  • Each type of WT1-expressing cancer or tumor, and cancer or tumor suspected to express WT1, represents a separate embodiment of the present invention.
  • multiple peptides of this invention are used to stimulate an immune response in methods of the present invention.
  • the methods disclosed herein will be understood by those in the art to enable design of other WT1-derived peptides.
  • the methods further enable design of peptides binding to other HLA molecules.
  • the methods further enable design of vaccines combining WT1-derived peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
  • vaccines of the present invention have the advantage of activating or eliciting WT1-specific CD4 + T cells containing a variety of different HLA class II alleles.
  • the vaccines have the advantage of activating or eliciting WT1-specific CD4 + T cells in a substantial proportion of the population (e.g. in different embodiments, 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%, 90%, 95%, or greater than 95%).
  • the vaccines activate or elicit WT1-specific CD4 + T cells in a substantial proportion of a particular population (e.g. American Caucasians). Each possibility represents a separate embodiment of the present invention.
  • methods of the present invention provide for an improvement in an immune response that has already been mounted by a subject.
  • methods of the present invention comprise administering the peptide, composition, or vaccine 2 or more times.
  • the peptides are varied in their composition, concentration, or a combination thereof.
  • the peptides provide for the initiation of an immune response against an antigen of interest in a subject who has not yet initiated an immune response against the antigen.
  • reference to modulation of the immune response involves, either or both the humoral and cell-mediated arms of the immune system, which is accompanied by the presence of Th2 and Th1 T helper cells, respectively, or in another embodiment, each arm individually.
  • the methods affecting the growth of a tumor result in (1) the direct inhibition of tumor cell division, or (2) immune cell mediated tumor cell lysis, or both, which leads to a suppression in the net expansion of tumor cells.
  • tumor inhibition is determined by measuring the actual tumor size over a period of time.
  • tumor inhibition can be determined by estimating the size of a tumor (over a period of time) utilizing methods well known to those of skill in the art. More specifically, a variety of radiologic imaging methods (e.g., single photon and positron emission computerized tomography; see generally, “Nuclear Medicine in Clinical Oncology,” Winkler, C. (ed.) Springer-Verlag, New York, 1986), can be utilized to estimate tumor size.
  • radiologic imaging methods e.g., single photon and positron emission computerized tomography; see generally, “Nuclear Medicine in Clinical Oncology,” Winkler, C. (ed.) Springer-Verlag, New York, 1986
  • imaging agents can also utilize a variety of imaging agents, including for example, conventional imaging agents (e.g., Gallium-67 citrate), as well as specialized reagents for metabolite imaging, receptor imaging, or immunologic imaging (e.g., radiolabeled monoclonal antibody specific tumor markers).
  • conventional imaging agents e.g., Gallium-67 citrate
  • immunologic imaging e.g., radiolabeled monoclonal antibody specific tumor markers
  • non-radioactive methods such as ultrasound (see, “Ultrasonic Differential Diagnosis of Tumors”, Kossoff and Fukuda, (eds.), Igaku-Shoin, New York, 1984), can also be utilized to estimate the size of a tumor.
  • in vitro methods can be utilized in order to predict in vivo tumor inhibition.
  • Representative examples include lymphocyte mediated anti-tumor cytolytic activity determined for example, by a 51 Cr release assay (Examples), tumor dependent lymphocyte proliferation (Ioannides, et al., J. Immunol. 146(5):1700-1707, 1991), in vitro generation of tumor specific antibodies (Herlyn, et al., J. Immunol. Meth.
  • cell e.g., CTL, helper T-cell
  • humoral e.g., antibody
  • methods of suppressing tumor growth indicate a growth state that is curtailed compared to growth without contact with, or exposure to a peptide of this invention.
  • Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a 3 H-thymidine incorporation assay, or counting tumor cells. “Suppressing” tumor cell growth refers, in other embodiments, to slowing, delaying, or stopping tumor growth, or to tumor shrinkage. Each possibility represents a separate embodiment of the present invention.
  • WT1 expression is measured.
  • WT1 transcript expression is measured.
  • WT1 protein levels in the tumor are measured.
  • lymphocyte proliferation assays wherein T cell uptake of a radioactive substance, e.g. 3 H-thymidine is measured as a function of cell proliferation.
  • detection of T cell proliferation is accomplished by measuring increases in interleukin-2 (IL-2) production, Ca 2+ flux, or dye uptake, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium.
  • IL-2 interleukin-2
  • dye uptake such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium.
  • CTL stimulation is determined by means known to those skilled in the art, including, detection of cell proliferation, cytokine production and others.
  • Analysis of the types and quantities of cytokines secreted by T cells upon contacting ligand-pulsed targets can be a measure of functional activity.
  • Cytokines can be measured by ELISA or ELISPOT assays to determine the rate and total amount of cytokine production. (Fujihashi K. et al. (1993) J. Immunol. Meth. 160: 181; Tanguay S. and Killion J. J. (1994) Lymphokine Cytokine Res. 13:259).
  • CTL activity is determined by 51 Cr-release lysis assay. Lysis of peptide-pulsed 51 Cr-labeled targets by antigen-specific T cells can be compared for target cells pulsed with control peptide.
  • T cells are stimulated with a peptide of this invention, and lysis of target cells expressing the native peptide in the context of MHC can be determined. The kinetics of lysis as well as overall target lysis at a fixed timepoint (e.g., 4 hours) are used, in another embodiment, to evaluate ligand performance (Ware C. F. et al. (1983) J Immunol 131: 1312).
  • affinity is determined by TAP stabilization assays.
  • affinity is determined by competition radioimmunoassay.
  • the following protocol is utilized: Target cells are washed two times in PBS with 1% bovine serum albumin (BSA; Fisher Chemicals, Fairlawn, N.J.). Cells are resuspended at 10 7 /ml on ice, and the native cell surface bound peptides are stripped for 2 minutes at 0[deg.] C using citrate-phosphate buffer in the presence of 3 mg/ml beta2 microglobulin.
  • BSA bovine serum albumin
  • the pellet is resuspended at 5 ⁇ 10 6 cells/ml in PBS/1% BSA in the presence of 3 mg/ml beta2 microglobulin and 30 mg/ml deoxyribonuclease, and 200 ml aliquots are incubated in the presence or absence of HLA-specific peptides for 10 min at 20° C., then with 125 I-labeled peptide for 30 min at 20° C. Total bound 125 I is determined after two washes with PBS/2% BSA and one wash with PBS. Relative affinities are determined by comparison of escalating concentrations of the test peptide versus a known binding peptide.
  • a specificity analysis of the binding of peptide to HLA on surface of live cells is conducted to confirm that the binding is to the appropriate HLA molecule and to characterize its restriction.
  • This assay is performed, in another embodiment, on live fresh or 0.25% paraformaldehyde-fixed human PBMC, leukemia cell lines and EBV-transformed T-cell lines of specific HLA types.
  • an HLA class II-binding peptide of methods and compositions of the present invention is longer than the minimum length for binding to an HLA class II molecule, which is, in another embodiment, about 12 AA.
  • increasing the length of the HLA class II-binding peptide enables binding to more than one HLA class II molecule.
  • increasing the length enables binding to an HLA class II molecule whose binding motif is not known.
  • increasing the length enables binding to an HLA class I molecule.
  • the binding motif of the HLA class I molecule is known.
  • the binding motif of the HLA class I molecule is not known. Each possibility represents a separate embodiment of the present invention.
  • the peptides utilized in methods and compositions of the present invention comprise a non-classical amino acid such as: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991) J. Am Chem. Soc. 113:2275-2283); (2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine (Kazmierski and Hruby (1991) Tetrahedron Lett.
  • a non-classical amino acid such as: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991) J. Am Chem. Soc. 113:2275-2283); (2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine,
  • a peptide of this invention comprises an AA analog or peptidomimetic, which, in other embodiments, induces or favors specific secondary structures.
  • Such peptides comprise, in other embodiments, the following: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a [beta]-turn inducing dipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838); [beta]-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5081-5082); [beta]-turn inducing analogs (Kemp et al.
  • a peptide of this invention is conjugated to one of various other molecules, as described hereinbelow, which can be via covalent or non-covalent linkage (complexed), the nature of which varies, in another embodiment, depending on the particular purpose.
  • the peptide is covalently or non-covalently complexed to a macromolecular carrier, (e.g. an immunogenic carrier), including, but not limited to, natural and synthetic polymers, proteins, polysaccharides, polypeptides (amino acids), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids.
  • a peptide of this invention is linked to a substrate.
  • the peptide is conjugated to a fatty acid, for introduction into a liposome (U.S. Pat. No. 5,837,249).
  • a peptide of the invention is complexed covalently or non-covalently with a solid support, a variety of which are known in the art.
  • linkage of the peptide to the carrier, substrate, fatty acid, or solid support serves to increase an elicited an immune response.
  • the carrier is thyroglobulin, an albumin (e.g. human serum albumin), tetanus toxoid, polyamino acids such as poly (lysine: glutamic acid), an influenza protein, hepatitis B virus core protein, keyhole limpet hemocyanin, an albumin, or another carrier protein or carrier peptide; hepatitis B virus recombinant vaccine, or an APC.
  • albumin e.g. human serum albumin
  • tetanus toxoid polyamino acids such as poly (lysine: glutamic acid), an influenza protein, hepatitis B virus core protein, keyhole limpet hemocyanin, an albumin, or another carrier protein or carrier peptide
  • APC hepatitis B virus recombinant vaccine
  • amino acid refers to a natural or, in another embodiment, an unnatural or synthetic AA, and can include, in other embodiments, glycine, D- or L optical isomers, AA analogs, peptidomimetics, or combinations thereof.
  • cancer in another embodiment, the terms “cancer,” “neoplasm,” “neoplastic” or “tumor,” are used interchangeably and refer to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • the definition of a cancer cell includes not only a primary cancer cell, but also any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a tumor is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation, and in another embodiment, is identified by biochemical or immunologic findings, the latter which is used to identify cancerous cells, as well, in other embodiments.
  • peptides of this invention are synthesized using an appropriate solid-state synthetic procedure (see for example, Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco, Calif. (1968); Merrifield (1967) Recent Progress in Hormone Res 23: 451). The activity of these peptides is tested, in other embodiments, using assays as described herein.
  • the peptides of this invention are purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • immuno-affinity chromatography is used, whereby an epitope is isolated by binding it to an affinity column comprising antibodies that were raised against that peptide, or a related peptide of the invention, and were affixed to a stationary support.
  • affinity tags such as hexa-His (Invitrogen), Maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991) Meth. Enzymol. 194:508-509), glutathione-S-transferase, or others, are attached to the peptides of this invention to allow easy purification by passage over an appropriate affinity column.
  • Isolated peptides can also be physically characterized, in other embodiments, using such techniques as proteolysis, nuclear magnetic resonance, and x-ray crystallography.
  • the peptides of this invention are produced by in vitro translation, through known techniques, as will be evident to one skilled in the art.
  • the peptides are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand, (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320).
  • the peptides of this invention further comprise a detectable label, which in another embodiment, is fluorescent, or in another embodiment, luminescent, or in another embodiment, radioactive, or in another embodiment, electron dense.
  • the detectable label comprises, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-galactosidase, luciferase, 32 P, 125 I, 3 H and 14 C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone, luciferin or any number of other such labels known to one skilled in the art.
  • the particular label used will depend upon the type of immunoassay used.
  • a peptide of this invention is linked to a substrate, which, in another embodiment, serves as a carrier. In another embodiment, linkage of the peptide to a substrate serves to increase an elicited an immune response.
  • peptides of this invention are linked to other molecules, as described herein, using conventional cross-linking agents such as carbodiimide.
  • carbodiimide are 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and 1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.
  • the cross-linking agents comprise cyanogen bromide, glutaraldehyde and succinic anhydride.
  • any of a number of homo-bifunctional agents including a homo-bifunctional aldehyde, a homo-bifunctional epoxide, a homo-bifunctional imido-ester, a homo-bifunctional N-hydroxysuccinimide ester, a homo-bifunctional maleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyl disulfide, a homo-bifunctional aryl halide, a homo-bifunctional hydrazide, a homo-bifunctional diazonium derivative and a homo-bifunctional photoreactive compound can be used.
  • hetero-bifunctional compounds for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine-reactive and a photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl-reactive group.
  • the homo-bifunctional cross-linking agents include the bifunctional N-hydroxysuccinimide esters dithiobis(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imido-esters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers 1,4-di-[3′-(2′-pyridyldithio)propionamido]butane, bismaleimidohexane, and bis-N-maleimido-1,8-octane; the bifunctional aryl halides 1,5-difluoro-2,4-dinitrobenzene and 4,4′-difluoro-3,3′-dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[b-(4-azidosalicylamid
  • hetero-bifunctional cross-linking agents used to link the peptides to other molecules include, but are not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MB S (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS (N-(.gamma-maleimidobutyryloxy)succmimide ester), MPBH (4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N-maleimidomethyl) cyclohexane-1-carboxyl-hydrazide), SMCC (succinimidyl-4
  • the peptides of the invention are formulated as non-covalent attachment of monomers through ionic, adsorptive, or biospecific interactions.
  • Complexes of peptides with highly positively or negatively charged molecules can be accomplished, in another embodiment, through salt bridge formation under low ionic strength environments, such as in deionized water. Large complexes can be created, in another embodiment, using charged polymers such as poly-(L-glutamic acid) or poly-(L-lysine), which contain numerous negative and positive charges, respectively.
  • peptides are adsorbed to surfaces such as microparticle latex beads or to other hydrophobic polymers, of D-biotin (NHS-biotin), which reacts with available amine groups.
  • a peptide of the present invention is linked to a carrier.
  • the carrier is KLH.
  • the carrier is any other carrier known in the art, including, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly (lysine:glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. Each possibility represents a separate embodiment of the present invention.
  • the peptides of this invention are conjugated to a lipid, such as P3 CSS. In another embodiment, the peptides of this invention are conjugated to a bead.
  • compositions of this invention further comprise immunomodulating compounds.
  • the immunomodulating compound is a cytokine, chemokine, or complement component that enhances expression of immune system accessory or adhesion molecules, their receptors, or combinations thereof.
  • the immunomodulating compound include interleukins, for example interleukins 1 to 15, interferons alpha, beta or gamma, tumour necrosis factor, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, macrophage inflammatory peptides MIP-Ia and MIP-Ib, complement components, or combinations thereof.
  • interleukins for example interleukins 1 to 15, interferons alpha, beta or gamma, tumour necrosis factor, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage chemo
  • the immunomodulating compound stimulate expression, or enhanced expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-1, 2 or 3, cytokine receptors, or combination thereof.
  • the immunomodulatory compound induces or enhances expression of co-stimulatory molecules that participate in the immune response, which include, in some embodiments, CD40 or its ligand, CD28, CTLA-4 or a B7 molecule.
  • the immunomodulatory compound induces or enhances expression of a heat stable antigen (HSA) (Liu Y. et al. (1992) J. Exp. Med. 175:437-445), chondroitin sulfate-modified MHC invariant chain (Ii-CS) (Naujokas M. F. et al (1993) Cell 74:257-268), or an intracellular adhesion molecule 1 (ICAM-I) (Van R. H. (1992) Cell 71: 1065-1068), which assists, in another embodiment, co-stimulation by interacting with their cognate ligands on the T cells.
  • HSA heat stable antigen
  • Ii-CS chondroitin sulfate-modified MHC invariant chain
  • the composition comprises a solvent, including water, dispersion media, cell culture media, isotonic agents and the like.
  • the solvent is an aqueous isotonic buffered solution with a pH of around 7.0.
  • the composition comprises a diluent such as water, phosphate buffered saline, or saline.
  • the composition comprises a solvent, which is non-aqueous, such as propyl ethylene glycol, polyethylene glycol and vegetable oils.
  • composition is formulated for administration by any of the many techniques known to those of skill in the art.
  • this invention provides for administration of the pharmaceutical composition parenterally, intravenously, subcutaneously, intradermally, intramucosally, topically, orally, or by inhalation.
  • the vaccine comprising a peptide of this invention further comprises a cell population, which, in another embodiment, comprises lymphocytes, monocytes, macrophages, dendritic cells, endothelial cells, stem cells or combinations thereof, which, in another embodiment are autologous, syngeneic or allogeneic, with respect to each other.
  • the cell population comprises a peptide of the present invention.
  • the cell population takes up the peptide.
  • the cell populations of this invention are obtained from in vivo sources, such as, for example, peripheral blood, leukopheresis blood product, apheresis blood product, peripheral lymph nodes, gut associated lymphoid tissue, spleen, thymus, cord blood, mesenteric lymph nodes, liver, sites of immunologic lesions, e.g. synovial fluid, pancreas, cerebrospinal fluid, tumor samples, granulomatous tissue, or any other source where such cells can be obtained.
  • the cell populations are obtained from human sources, which are, in other embodiments, from human fetal, neonatal, child, or adult sources.
  • the cell populations of this invention are obtained from animal sources, such as, for example, porcine or simian, or any other animal of interest. In another embodiment, the cell populations of this invention are obtained from subjects that are normal, or in another embodiment, diseased, or in another embodiment, susceptible to a disease of interest.
  • the cell populations of this invention are separated via affinity-based separation methods.
  • Techniques for affinity separation include, in other embodiments, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or use in conjunction with a monoclonal antibody, for example, complement and cytotoxins, and “panning” with an antibody attached to a solid matrix, such as a plate, or any other convenient technique.
  • separation techniques include the use of fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • any technique that enables separation of the cell populations of this invention can be employed, and is to be considered as part of this invention.
  • the dendritic cells are from the diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues, qualified as such (Steinman (1991) Ann. Rev. Immunol. 9:271-296).
  • the dendritic cells used in this invention are isolated from bone marrow, or in another embodiment, derived from bone marrow progenitor cells, or, in another embodiment, from isolated from/derived from peripheral blood, or in another embodiment, derived from, or are a cell line.
  • the cell populations described herein are isolated from the white blood cell fraction of a mammal, such as a murine, simian or a human (See, e.g., WO 96/23060).
  • the white blood cell fraction can be, in another embodiment, isolated from the peripheral blood of the mammal.
  • the DC are isolated via a method which includes the following steps: (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the art such as leukophoresis; (b) separating the white blood cell fraction of step (a) into four or more subfractions by countercurrent centrifugal elutriation; (c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSF and IL-4, (d) identifying the dendritic cell-enriched fraction from step (c); and (e) collecting the enriched fraction of step (d), preferably at about 4[deg.] C.
  • the dendritic cell-enriched fraction is identified by fluorescence-activated cell sorting, which identifies at least one of the following markers: HLA-DR, HLA-DQ, or B7.2, and the simultaneous absence of the following markers: CD3, CD14, CD16, 56, 57, and CD 19, 20.
  • the cell population comprises lymphocytes, which are, in another embodiment, T cells, or in another embodiment, B cells.
  • the T cells are, in other embodiments, characterized as NK cells, helper T cells, cytotoxic T lymphocytes (CTL), TBLs, native T cells, or combinations thereof. It is to be understood that T cells which are primary, or cell lines, clones, etc. are to be considered as part of this invention.
  • the T cells are CTL, or CTL lines, CTL clones, or CTLs isolated from tumor, inflammatory, or other infiltrates.
  • hematopoietic stem or early progenitor cells comprise the cell populations used in this invention.
  • such populations are isolated or derived, by leukaphoresis.
  • the leukapheresis follows cytokine administration, from bone marrow, peripheral blood (PB) or neonatal umbilical cord blood.
  • the stem or progenitor cells are characterized by their surface expression of the surface antigen marker known as CD34 + , and exclusion of expression of the surface lineage antigen markers, Lin ⁇ .
  • the subject is administered a peptide, composition or vaccine of this invention, in conjunction with bone marrow cells.
  • the administration together with bone marrow cells embodiment follows previous irradiation of the subject, as part of the course of therapy, in order to suppress, inhibit or treat cancer in the subject.
  • the phrase “contacting a cell” or “contacting a population” refers to a method of exposure, which can be, in other embodiments, direct or indirect.
  • such contact comprises direct injection of the cell through any means well known in the art, such as microinjection.
  • supply to the cell is indirect, such as via provision in a culture medium that surrounds the cell, or administration to a subject, via any route well known in the art, and as described herein.
  • CTL generation of methods of the present invention is accomplished in vivo, and is effected by introducing into a subject an antigen presenting cell contacted in vitro with a peptide of this invention (See for example Paglia et al. (1996) J. Exp. Med. 183:317-322).
  • the peptides of methods and compositions of the present invention are delivered to APC.
  • the peptide-pulsed APC are administered to a subject to elicit and immune response or treat or inhibit growth or recurrence of a tumor.
  • Each possibility represents a separate embodiment of the present invention.
  • the peptides are delivered to APC in the form of cDNA encoding the peptides.
  • the term “antigen-presenting cells” refers to dendritic cells (DC), monocytes/macrophages, B lymphocytes or other cell type(s) expressing the necessary MHC/co-stimulatory molecules, which effectively allow for T cell recognition of the presented peptide.
  • the APC is a cancer cell. Each possibility represents a separate embodiment of the present invention.
  • the CTL are contacted with 2 or more APC populations.
  • the 2 or more APC populations present different peptides. Each possibility represents a separate embodiment of the present invention.
  • techniques that lead to the expression of antigen in the cytosol of APC are used to deliver the peptides to the APC.
  • Methods for expressing antigens on APC are well known in the art.
  • the techniques include (1) the introduction into the APC of naked DNA encoding a peptide of this invention, (2) infection of APC with recombinant vectors expressing a peptide of this invention, and (3) introduction of a peptide of this invention into the cytosol of an APC using liposomes.
  • foster APC such as those derived from the human cell line 174 ⁇ CEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763-1771), are used, as exemplified herein.
  • the subject is exposed to a peptide, or a composition/cell population comprising a peptide of this invention, which differs from the native protein expressed, wherein subsequently a host immune cross-reactive with the native protein/antigen develops.
  • the subject as referred to in any of the methods or embodiments of this invention is a human.
  • the subject is a mammal, which can be a mouse, rat, rabbit, hamster, guinea pig, horse, cow, sheep, goat, pig, cat, dog, monkey, or ape. Each possibility represents a separate embodiment of the present invention.
  • peptides, vaccines, and compositions of this invention stimulate an immune response that results in tumor cell lysis.
  • any of the methods described herein is used to elicit CTL, which are elicited in vitro.
  • the CTL are elicited ex-vivo.
  • the CTL are elicited in vitro.
  • the resulting CTL are, in another embodiment, administered to the subject, thereby treating the condition associated with the peptide, an expression product comprising the peptide, or a homologue thereof.
  • the method entails introduction of the genetic sequence that encodes the peptides of this invention using, e.g., one or more nucleic acid delivery techniques.
  • Nucleic acids of the invention include, in another embodiment, DNA, RNA and mixtures of DNA and RNA, alone or in conjunction with non-nucleic acid components.
  • the method comprises administering to the subject a vector comprising a nucleotide sequence, which encodes a peptide of the present invention (Tindle, R. W. et al. Virology (1994) 200:54).
  • the method comprises administering to the subject naked DNA which encodes a peptide, or in another embodiment, two or more peptides of this invention (Nabel, et al. PNAS-USA (1990) 90: 11307).
  • label, et al. PNAS-USA (1990) 90: 11307 In another embodiment, multi-epitope, analogue-based cancer vaccines are utilized (Fikes et al, Design of multi-epitope, analogue-based cancer vaccines. Expert Opin Biol Ther. 2003 September; 3(6):985-93). Each possibility represents a separate embodiment of the present invention.
  • Nucleic acids can be administered to a subject via any means as is known in the art, including parenteral or intravenous administration, or in another embodiment, by means of a gene gun. In another embodiment, the nucleic acids are administered in a composition, which correspond, in other embodiments, to any embodiment listed herein.
  • Vectors for use according to methods of this invention can comprise any vector that facilitates or allows for the expression of a peptide of this invention.
  • Vectors comprise, in some embodiments, attenuated viruses, such as vaccinia or fowlpox, such as described in, e.g., U.S. Pat. No. 4,722,848, incorporated herein by reference.
  • the vector is BCG (Bacille Calmette Guerin), such as described in Stover et al. (Nature 351:456-460 (1991)).
  • BCG Bacille Calmette Guerin
  • Salmonella typhi vectors and the like will be apparent to those skilled in the art from the description herein.
  • the vector further encodes for an immunomodulatory compound, as described herein.
  • the subject is administered an additional vector encoding same, concurrent, prior to or following administration of the vector encoding a peptide of this invention to the subject.
  • the peptides, compositions and vaccines of this invention are administered to a subject, or utilized in the methods of this invention, in combination with other anticancer compounds and chemotherapeutics, including monoclonal antibodies directed against alternate cancer antigens, or, in another embodiment, epitopes that consist of an AA sequence which corresponds to, or in part to, that from which the peptides of this invention are derived.
  • the dosage is 20 ⁇ g per peptide per day. In another embodiment, the dosage is 10 ⁇ g/peptide/day. In another embodiment, the dosage is 30 ⁇ g/peptide/day. In another embodiment, the dosage is 40 ⁇ g/peptide/day. In another embodiment, the dosage is 60 ⁇ g/peptide/day. In another embodiment, the dosage is 80 ⁇ g/peptide/day. In another embodiment, the dosage is 100 ⁇ g/peptide/day. In another embodiment, the dosage is 150 ⁇ g/peptide/day. In another embodiment, the dosage is 200 ⁇ g/peptide/day. In another embodiment, the dosage is 300 ⁇ g/peptide/day.
  • the dosage is 400 ⁇ g/peptide/day. In another embodiment, the dosage is 600 ⁇ g/peptide/day. In another embodiment, the dosage is 800 ⁇ g/peptide/day. In another embodiment, the dosage is 1000 ⁇ g/peptide/day. In another embodiment, the dosage is 1500 ⁇ g/peptide/day. In another embodiment, the dosage is 2000 ⁇ g/peptide/day.
  • the dosage is 10 ⁇ g/peptide/dose. In another embodiment, the dosage is 30 ⁇ g/peptide/dose. In another embodiment, the dosage is 40 ⁇ g/peptide/dose. In another embodiment, the dosage is 60 ⁇ g/peptide/dose. In another embodiment, the dosage is 80 ⁇ g/peptide/dose. In another embodiment, the dosage is 100 ⁇ g/peptide/dose. In another embodiment, the dosage is 150 ⁇ g/peptide/dose. In another embodiment, the dosage is 200 ⁇ g/peptide/dose. In another embodiment, the dosage is 300 ⁇ g/peptide/dose. In another embodiment, the dosage is 400 ⁇ g/peptide/dose. In another embodiment, the dosage is 600 ⁇ g/peptide/dose.
  • the dosage is 800 ⁇ g/peptide/dose. In another embodiment, the dosage is 1000 ⁇ g/peptide/dose. In another embodiment, the dosage is 1500 ⁇ g/peptide/dose. In another embodiment, the dosage is 2000 ⁇ g/peptide/dose.
  • the dosage is 10-20 ⁇ g/peptide/dose. In another embodiment, the dosage is 20-30 ⁇ g/peptide/dose. In another embodiment, the dosage is 20-40 ⁇ g/peptide/dose. In another embodiment, the dosage is 30-60 ⁇ g/peptide/dose. In another embodiment, the dosage is 40-80 ⁇ g/peptide/dose. In another embodiment, the dosage is 50-100 ⁇ g/peptide/dose. In another embodiment, the dosage is 50-150 ⁇ g/peptide/dose. In another embodiment, the dosage is 100-200 ⁇ g/peptide/dose. In another embodiment, the dosage is 200-300 ⁇ g/peptide/dose. In another embodiment, the dosage is 300-400 ⁇ g/peptide/dose.
  • the dosage is 400-600 ⁇ g/peptide/dose. In another embodiment, the dosage is 500-800 ⁇ g/peptide/dose. In another embodiment, the dosage is 800-1000 ⁇ g/peptide/dose. In another embodiment, the dosage is 1000-1500 ⁇ g/peptide/dose. In another embodiment, the dosage is 1500-2000 ⁇ g/peptide/dose.
  • the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts.
  • the dosage is 20 mg per peptide per day. In another embodiment, the dosage is 10 mg/peptide/day. In another embodiment, the dosage is 30 mg/peptide/day. In another embodiment, the dosage is 40 mg/peptide/day. In another embodiment, the dosage is 60 mg/peptide/day. In another embodiment, the dosage is 80 mg/peptide/day. In another embodiment, the dosage is 100 mg/peptide/day. In another embodiment, the dosage is 150 mg/peptide/day. In another embodiment, the dosage is 200 mg/peptide/day. In another embodiment, the dosage is 300 mg/peptide/day. In another embodiment, the dosage is 400 mg/peptide/day. In another embodiment, the dosage is 600 mg/peptide/day. In another embodiment, the dosage is 800 mg/peptide/day. In another embodiment, the dosage is 1000 mg/peptide/day.
  • the dosage is 10 mg/peptide/dose. In another embodiment, the dosage is 30 mg/peptide/dose. In another embodiment, the dosage is 40 mg/peptide/dose. In another embodiment, the dosage is 60 mg/peptide/dose. In another embodiment, the dosage is 80 mg/peptide/dose. In another embodiment, the dosage is 100 mg/peptide/dose. In another embodiment, the dosage is 150 mg/peptide/dose. In another embodiment, the dosage is 200 mg/peptide/dose. In another embodiment, the dosage is 300 mg/peptide/dose. In another embodiment, the dosage is 400 mg/peptide/dose. In another embodiment, the dosage is 600 mg/peptide/dose. In another embodiment, the dosage is 800 mg/peptide/dose. In another embodiment, the dosage is 1000 mg/peptide/dose.
  • the dosage is 10-20 mg/peptide/dose. In another embodiment, the dosage is 20-30 mg/peptide/dose. In another embodiment, the dosage is 20-40 mg/peptide/dose. In another embodiment, the dosage is 30-60 mg/peptide/dose. In another embodiment, the dosage is 40-80 mg/peptide/dose. In another embodiment, the dosage is 50-100 mg/peptide/dose. In another embodiment, the dosage is 50-150 mg/peptide/dose. In another embodiment, the dosage is 100-200 mg/peptide/dose. In another embodiment, the dosage is 200-300 mg/peptide/dose. In another embodiment, the dosage is 300-400 mg/peptide/dose. In another embodiment, the dosage is 400-600 mg/peptide/dose. In another embodiment, the dosage is 500-800 mg/peptide/dose. In another embodiment, the dosage is 800-1000 mg/peptide/dose.
  • the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts.
  • the present invention provides a kit comprising a peptide, composition or vaccine of the present invention.
  • the kit further comprises a label or packaging insert.
  • the kit is used for detecting a WT1-specific CD4 response through the use of a delayed-type hypersensitivity test.
  • the kit is used for any other method enumerated herein.
  • the kit is used for any other method known in the art. Each possibility represents a separate embodiment of the present invention.
  • WT1 is considered one of the most promising (47).
  • the number of immunogenic WT1 peptide antigens previously identified and reported is very limited, and largely confined to a set of peptides presented by the HLA alleles A0201, A2402 and DRB10401.
  • WT1 peptide-specific IFN ⁇ +CD4 + and CD8 T-cell responses were generated from the blood of 41/56 (78%) normal donors, and thereafter the epitopes eliciting these responses and their presenting HLA alleles were identified.
  • the new immunogenic peptides identified include 36 peptides presented by class I HLA alleles and 5 presented by class II HLA alleles.
  • T-cell lines specific for 29 epitopes including 2/4 epitopes presented by class II and 27/32 presented by class I alleles, were also able to lyse WT1+ leukemic blasts sharing the T-cells' restricting HLA allele.
  • FIG. 4 maps are shown of the WT1 protein.
  • FIG. 4C defines the localization of all previously reported antigenic epitopes presented by HLA class I and II alleles;
  • FIG. 4D depicts the location of immunogenic peptides identified in this report.
  • the 11 epitopes previously reported to be presented by class I and 10 presented by class II HLA alleles are principally clustered in sequences encoded by exons 1, 7 and 10
  • the epitopes recognized by normal T-cells sensitized with the WT1 peptide pool are principally clustered in sequences encoded by the first 5 exons.
  • 26 of the new epitopes are included in each of the four major isoforms of WT1 resulting from splice variants that do or do not include the 17 amino acid sequence (aas 250-266) in exon 5 or the three amino acid sequence ( 400-410 KTS) between zinc fingers 3 and 4. While the epitopes are broadly distributed, clusters of epitopes were detected in the RNA recognition domain in exon 1 and the activation domain (aa 181-250) ( FIG. 4F ) proximal to the spliced 17aa segment in exon 5. The latter area also contained those epitopes most frequently recognized by multiple donors ( FIG. 4E ).
  • epitopes map to a 126 amino acid sequence at the N terminus encoded by a segment of the WT1 gene initially described by Gessler et al (37) that is centromeric to exon 1 of the (Exon 5+, KTS+) isoform of WT1 and includes the long isoform of WT1 initiated at a CUG codon upstream of the AUG initiator for exon 1.50 Strikingly, each of the epitopes identified in this sequence elicits IFN ⁇ + T-cells that are cytolytic against leukemic blasts coexpressing WT1 and the T-cells' restricting HLA allele.
  • WT1 and MART-1 differentially expressed by specific tumors
  • WT1 and MART-1 have been shown to elicit responses in normal donors (31,32,51-54).
  • T-cells specific for each of these proteins have been recorded in a proportion of patients with tumors overexpressing them (55).
  • T-cells specific for the RMF and CMT peptides of WT1 have been detected in patients with leukemias, myeloma, carcinoma of the breast and prostate and other solid tumors (31,32,56-61).
  • Responses to several of the WT1 epitopes identified in the present study in 50-60% of patients with ovarian cancer have been documented.
  • peptides from the WT1 protein sequence that are upstream from exon 1, i.e., within the first 126 amino acids of SEQ ID NO:194, are heretofore unrecognized sites of immunogenic epitopes and therefore peptides useful for the purposes herein.
  • Peptides were synthesized by Genemed Synthesis Inc, CA using fluorenylmethoxycarbonyl chemistry and solid phase synthesis, and were purified by high pressure liquid chromatography (HPLC). The quality of the peptides was assessed by HPLC analysis, and the expected molecular weight was measured using matrix-assisted laser desorption mass spectrometry. Peptides were sterile and >90% pure. The peptides were dissolved in DMSO and diluted in PBS at pH 7.4 or saline solution to yield a concentration of 5 milligrams per milliliter (mg/ml) and were stored at ⁇ 80° C. For in vitro experiments, an irrelevant control peptide, HLA A24 consensus, was used.
  • Irrelevant control peptides used in in vitro experiments were: RAS (TEYKLVVVGAPGVGKSALTIQ; SEQ ID No: 198) or CML b2a2 (VHSIPLTINKEEALQRPVASDFE; SEQ ID No: 199) for Class II, and HIV pol (ILKEPVHGV; SEQ ID No: 200) or CML F (YLKALQRPY; SEQ ID No: 201) for Class I.
  • T2 is a human cell line lacking TAP1 and TAP2 and therefore unable to present peptides derived from cytosolic proteins.
  • Raji cells are a human Burkitt lymphoma cells that exhibit a high level of TAP expression.
  • H-Meso1A NCI, Bethesda, Md.
  • JMN and VAMT Dr. Sirotnak, Memorial Sloan Kettering Cancer Center (MSKCC)
  • H-2452 and H2373 Dr. Pass, Karmanos Cancer Institute, Wayne State University, Detroit, Mich.
  • H28 and MSTO American Type Culture Collection (ATCC, Manassas, Va.).
  • ATCC Manassas, Va.
  • Mesothelioma cell lines Meso 11, Meso 34, Meso 37, Meso 47 and Meso 56 were obtained from Dr. M Gregoire (Institute of Biology, France) and cultured in RPMI 1640 (Life Technologies)+10% fetal calf serum (FCS), 1% penicillin—streptomycin, and 1% L-glutamine. All cells were HLA typed by the Department of Cellular Immunology at MSKCC. Melanoma cell line Mewo (WT1 ⁇ A201+) was obtained from the ATCC. SKRC-52 renal cell carcinoma was obtained from L. Old of the Ludwig Institute.
  • Leukemia cell lines were cultured in RPMI 1640+10% FCS, 1% penicillin-streptomycin, 2 mM glutamine and 2-mercaptoethanol at 37° C./5% CO2.
  • SKLY-16 is a human B cell lymphoma (WT1 ⁇ , A0201+); K562, RwLeu4 and HL60, all WT1+ leukemias, were obtained from the ATCC.
  • T2 assay for peptide binding and stabilization of HLA A0201 molecules T2 cells (TAP ⁇ , HLA-A0201 + ) were incubated overnight at 27° C. at a concentration of 1 ⁇ 10 6 cells/ml in FCS-free RPMI medium supplemented with 5 ⁇ g/ml human ⁇ 2m (Sigma, St Louis, Mo.) in the absence (negative control) or presence of either a positive reference tyrosinase peptide or test peptides at various final concentrations (50, 10, 1, and 0.1 micrograms ( ⁇ g)/ml). Following a 4-hour incubation with 5 ⁇ g/ml brefeldin A (Sigma), T2 cells were labeled for 30 minutes at 4° C.
  • MIF mean intensity of fluorescence
  • the number of stable peptide-HLA-A2.1 complexes was estimated as described above by immunofluorescence.
  • Peripheral blood was obtained from 56 consenting normal donors according to protocols approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (New York, N.Y.). All donors were typed for HLA-A, B, C, DR and DQ at high resolution by standard techniques.
  • Cytokine-activated monocytes were used as antigen presenting cells (APCs), and generated as previously described (32). Briefly, peripheral blood monocytes were separated by adherence on plastic and cultured in RPMI1640 containing 1% autologous serum. GM-CSF (Berlex, Montville, N.J.) and interleukin-4 (IL-4) (R&D Systems, Minneapolis, Minn.) were added to final concentrations of 2000 U/ml and 1000 U/ml respectively on days 0, 2, 4.
  • GM-CSF Stemas, Montville, N.J.
  • IL-4 interleukin-4
  • CAMs harvested on day 7 of culture expressed CD83, CD80, CD86, and HLA class I and II alleles as determined by FACS analysis.
  • EBV-BLCL were also used as WT1 peptide loaded and control APCs or as targets as specified in the experiments. They were generated by infection of peripheral blood mononuclear cells (PBMC) with EBV strain B95.8 (38,39) as previously described.
  • PBMC peripheral blood mononuclear cells
  • EBV transformed BLCL (EBV-BLCL) were cultured in RPMI1640(Gemini) with 10% fetal calf serum (Gemini) in the presence of Acyclovir.
  • PBMC peripheral blood mononuclear cells
  • CD56 pre-coated microbeads
  • Enriched T-cell fractions were stimulated at a 20:1 responder:stimulator ratio with autologous CAMs or EBV-BLCL that had been pre-loaded for 3 hours with the total pool of the WT1 pentadecapeptides in serum-free medium and irradiated to 3000 cGy.
  • T-cells were cultured in Yssel's medium supplemented with 5% AB human serum (YHS, Gemini), re-stimulated weekly with the autologous WT1 total pool-loaded CAMs or EBV-BLCL and fed with interleukin-2(IL-2) (Collaborative Biomedical Products, Bedford, Mass.) every 2-3 days at 10-50 U/ml.
  • YHS human serum
  • IL-2 Interleukin-2
  • WT1+ leukemias included blast cells from 11 primary AMLs, 3 primary ALLs and 1 B-cell precursor ALL cell line.
  • Ten WT1 ⁇ leukemias were used as controls, and included 3 B-cell precursor ALLs and 7 AMLs.
  • EBV BLCL and leukemia cells were typed for HLA A, B, C, DR and DQ alleles at high resolution by standard techniques.
  • the proportion and phenotype (CD4 and CD8) of T-cells generating IFN ⁇ in response to secondary stimulation with the WT1 total pool, WT1 subpools or single WT1 15-mer or 9-mer WT1 peptides loaded on autologous PBMC were measured by FACS analysis of T-cells containing intracellular IFN ⁇ as previously described (38,40).
  • the W-1-specific and HLA-restricted cytotoxic activity of sensitized T-cells was measured in standard Cr51 release assays against a panel of HLA-matched and mismatched CAM targets either unmodified or loaded with the total pool, the identified 15-mer, or the 9-mer or 11-mer epitope of WT1 eliciting T-cell responses, as previously described (32).
  • the restricting HLA allele presenting each immunogenic epitope was identified by measuring the cytotoxicity of the sensitized T-cells against a panel of allogeneic CAMs pre-loaded with the peptide, each sharing a single HLA allele expressed on the responding WT1-specific T-cells as previously described (41).
  • the cytotoxic activity of the WT1 epitope-specific CTLs against WT1 ⁇ and WT1+ leukemia cell lines or primary leukemic cells expressing the restricting HLA alleles was also assessed in this cytotoxicity assay Cr51 assay as previously described (32).
  • enriched T-cells separated from PBMC of groups of normal donors expressing one of a series of prevalent HLA alleles i.e. HLA-A0201, A0301, A2402, B0702
  • HLA-A0201, A0301, A2402, B0702 a series of prevalent HLA alleles which were previously identified as a presenter of a newly identified WT1 epitope
  • AAPC artificial antigen-presenting cells
  • the panel of AAPCs includes AAPCs expressing one of the following single HLA alleles: HLA A0201, A0101, A0301, A2402, B0702 or B0801, which were generated as previously described (42). After 35 days of co-culture of T-cells with the peptide-loaded AAPCs in the presence of IL2, CTLs were secondarily stimulated overnight with autologous PBMC loaded with the sensitizing peptide or an unrelated peptide and tested for their IFN ⁇ response.
  • the responses were registered as positive if the proportion of T-cells producing IFN ⁇ in response to the secondary stimulation with autologous PBMC loaded with the stimulating WT1 derived peptide exceeded the background proportion of IFN ⁇ T-cells incubated with PBMC alone by two fold or more.
  • T-cells generated from 38/56 donors also exhibited cytotoxic activity against autologous PHA blasts loaded with the WT1 total pool ( FIG. 1B ), including T-cells from 38 of the 41 donors that produced IFN ⁇ in response to secondary stimulation with the WT1 peptide pool.
  • IFN ⁇ response measured by FACS staining in different responder cell populations peripheral blood derived PBMC, pre-generated CTLs sensitized in vitro with the RMF peptide loaded on autologous CAM and pre-generated CTLs sensitized with the total pool of WT1 15-mers
  • autologous PBMC either unmodified or loaded with one of the following: RMF peptide, dominant epitopes of WT1 identified by the epitope mapping approach in the WT1-total pool sensitized CTL, WT1 total pool of the 141 pentadecapeptides; D.
  • Cytotoxic activity of the WT specific T cells generated in vitro by sensitization with autologous CAMs loaded with the RMF 9-mer or with the total pool of the WT1 15-mers was assessed against autologous WT1 negative targets(PHA activated blasts) and the same targets loaded with RMF peptide, the total pool of WT1 15-mers or the dominant WT1 epitope identified for the same T cell line.
  • the epitopes recognized by T-cells sensitized in vitro with the total pool of overlapping WT1 pentadecapeptides were identified by quantitating IFN ⁇ + T-cells responding to a mapping grid of subpools of WT1 15-mers formed so that any single 15-mer is shared by only 2 intersecting subpools ( FIG. 2B ). As shown for a representative example in FIG. 2C , significantly increased numbers of IFN ⁇ + T-cells are selectively generated in response to subpools #3 and #19 which share the pentadecapeptide #75. The T-cells were then stimulated with neighboring 15-mers, each overlapping peptide #75 by 11aa.
  • IFN ⁇ + T-cells are selectively generated in response to peptide #75 ( FIG. 2D ).
  • the newly identified immunogenic WT1 epitope is 174-182 HSFKHEDPM.
  • the cytotoxic activity of these T-cells was assessed against a panel of allogeneic CAMs either unmodified or loaded with this peptide, each sharing one HLA allele expressed by the tested CTLs.
  • the T-cells selectively lysed peptide loaded autologous targets and targets expressing the HLA-B3501 allele, and did not lyse peptide-loaded targets sharing other HLA alleles inherited by the T-cell donor.
  • These T-cells also lysed WT1+ BALL cells coexpressing the HLA-B3501 allele.
  • FIG. 2 Strategy for the generation of the total pool of overlapping pentadecapeptides spanning the whole sequence of the WT1 protein and epitope mapping: A. The sequence of the WT1 protein consisting of 575 amino acids and the principle of 11 amino acid overlapping pentadecapeptides are illustrated. A total of 141 pentadecapeptides are required to span the entire protein. The sequence of 575 aminoacids published by Gessler et al. (37), was employed. This sequence includes an additional 126 aminoacids in the N-terminus.
  • WT1 In order to match the sequential numbers of aminoacids within the WT1 sequence used with the longest, most frequently described WT1 isoform D we numbered the first 126aa with negative values and used the positive values to number the subsequent 449 aminoacids described in the longest isoform D;
  • B. The mapping grid consisting of 24 subpools each containing up to 12 WT1-derived pentadecapeptides. Each peptide is uniquely contained within two intersecting subpools: for example peptide 75 is uniquely shared by subpools 3 and 19;
  • C IFN ⁇ production by WT1 sensitized CTLs in response to secondary overnight stimulation with the subpools of WT1 pentadecapeptides loaded on autologous PBMC.
  • the CAMs or PHA blasts used in the assay are unmodified (grey bars) or loaded with the WT1 dominant epitope (black bars).
  • the WT1 specific cytotoxic activity of the WT1 CTLs is restricted by the B3501 HLA allele.
  • the immunogenic peptides of WT1 that were identified and their presenting HLA alleles are listed in Table 1. Of the 42 WT1 peptides eliciting T-cell responses, 41 are newly identified; only one of these WT1 peptides, the 126-134 RMFPNAPYL nonamer presented by HLA-A0201, has been previously described and shown to be immunogenic when presented by this allele (43) Peptide 91, 235-249 CMTWNQMNLGATLKG contains an epitope which, in the study, elicited CD4+ T-cell responses restricted by HLA DRB1 0402, but also contains the 235-243CMT nonamer known to be presented by HLA A0201 and HLA A2402 (29).
  • T-cell cultures exhibiting epitope-specific cytotoxic activity against peptide loaded targets, 36 could be tested for cytotoxic activity against WT1+ leukemic cells coexpressing the T-cell's restricting HLA allele. Of these 36, 27 exhibited HLA-restricted cytotoxic activity against the WT1+ leukemic cells (Table 2). T-cells specific for five peptides, 6-15 RDL, 46-54 SAY, 58-66 PAP, 225-233 NLY, and 436-445 NMH, presented by HLA A0201, could not lyse HLA-A0201 + WT1+ leukemic cells.
  • HLA B4001 restricted T-cells specific for the 46-54 SAY peptide, could lyse WT1+ leukemic coexpressing this HLA allele.
  • NMH peptide-specific HLA-restricted T-cell lines that lysed targets loaded with the NMH peptide coexpressing HLA A0201, B4001 or A2402 were only able to lyse WT1+ leukemic cells expressing the HLA B4001 allele.
  • the T-cells from each donor were sensitized with the identified epitope loaded on a panel of artificial antigen presenting cells (AAPC) (42) each expressing a single HLA allele, specifically A0201, A0301, A2402 or B0702.
  • AAPC artificial antigen presenting cells
  • the mapped peptide specificity i.e. ( ⁇ 99)-( ⁇ 91) THS, ( ⁇ 22)-( ⁇ 19) KLG, 22-31 GGC, 126-134 RMF and ( ⁇ 125)-( ⁇ 117) RQR) was identical to the peptide with the highest affinity for the presenting HLA allele predicted by the binding algorithm within the stimulating 15-mer.
  • the proportion of donors responding to individual mapped peptides were equal or greater than those generated in response to the neighboring epitopes predicted to have higher affinity.
  • IFN ⁇ + T-cell responses were generated to the 38-46 LDF peptide in 8/12 (67%) of HLA A0201 donors tested, while none responded to the predicted and previously reported (46) epitope 37-45 VLDFAPPGA Similarly, among HLA A2402+ donors, 4/6 donors (60%) responded to the 239-248 NQMNLGATL peptide while only 1 ⁇ 6 responded to the 235-243 CMTWNQMNL peptide previously reported to be presented by this allele (29).
  • the peptides were loaded on HLA A0201+ AAPCs and used to sensitize T-cells from 8 of the HLA A0201+ normal donors. After 35 days of sensitization, the T-cells were then washed and secondarily restimulated for 24 hours with aliquots of irradiated autologous PBMC loaded with each individual peptide. Responding IFN ⁇ + T-cells were then quantitated by FACS. The results, presented in FIG.
  • FIG. 5 presents maps of the WT1 protein.
  • FIG. 5C defines the localization of all previously reported antigenic epitopes presented by HLA class I and II alleles;
  • FIG. 5D depicts the location of immunogenic peptides identified in this report.
  • the 11 epitopes previously reported to be presented by class I and 10 presented by class II HLA alleles are principally clustered in sequences encoded by exons 1, 7 and 10, while the epitopes recognized by normal T-cells sensitized with the WT1 peptide pool are principally clustered in sequences encoded by the first 5 exons.
  • epitopes map to a 126 amino acid sequence at the N terminus encoded by a segment of the WT1 gene initially described by Gessler et a1 37 that is centromeric to exon 1 of the (Exon 5 + , KTS + ) isoform of WT1 and includes the long isoform of WT1 initiated at a CUG codon upstream of the AUG initiator for exon 1. 50 Strikingly, each of the epitopes identified in this sequence elicits IFN ⁇ + T-cells that are cytolytic against leukemic blasts coexpressing WT1 and the T-cells' restricting HLA allele.
  • the utility of peptides described herein in treating ovarian cancer was evaluated in two studies.
  • the inhibitory effect on ovarian tumor engraftment of T-cells specific for different WT1 peptides was evaluated by pre-incubating T cells at different doses with SKOV3-A2 ovarian carcinoma cells before injection into NOD/SCID mice.
  • T cell cultures specific for the following immunodominant epitopes were prepared using methods described above: A0201 restricted WT1 peptide LKTHTRTHT (SEQ ID NO:182) specific T cells; A0301 restricted WT1 peptide RQRPHPGAL (SEQ ID NO:142) specific T cells, and A0201 restricted WT1 peptide HFPPSLPPT (SEQ ID NO:145) T cells.
  • T cells to tumor cell ratios tested were 50:1, 10:1, 5:1 and control (no T cells). Following tumor injection, the tumor burden was monitored by bioluminescent imaging. For all three T cell lines at each dose, a significant reduction in tumor burden was observed over time vs. control.
  • mice survival was prolonged by pre-incubation of tumor cells with WT1 peptide specific T cells.
  • all mice were dead by 70 days post tumor injection.
  • Increased survival was seen dose-responsively with the T cell: tumor cell dose, which for the 50:1 dose for all T cell lines still had some animals alive at 96 days, and also at 10:1 for the LKTHTRTHT (SEQ ID NO:182) specific line.
  • WT1 peptide specific T cells were administered intravenously to NOD/SCID mice bearing pre-established ovarian carcinoma SKOV3-A2 xenografts.
  • T cell lines evaluated were: A0201 restricted WT1 peptide LKTHTRTHT (SEQ ID NO:182) specific T cells and A0301 restricted WT1 peptide RQRPHPGAL (SEQ ID NO:142) specific T cells.
  • Tumor burden was monitored by bioluminescence, tumor infiltration by human CD3+ cells evaluated and survival recorded. In both cases, the WT1 specific T cells afforded reduced tumor burden vs. control, increased tumor infiltration by human CD3+ cells and increased survival.
  • a phase I clinical trial was conducted using transplant donor-derived T-cells sensitized with the full pool of WT1 derived pentadecapeptides described above, in the adoptive therapy of patients who have relapsed following an allogeneic marrow transplant from a normal related or unrelated donor.
  • the HLA restricting alleles and corresponding immunodominant WT1 epitopes are as follows: A0201, SEQ ID NO:147; A0203, SEQ ID NOs:176 and 183; B3503 and C0401, SEQ ID NOs:161 and 169; A6901, SEQ ID NO:177; A0201, SEQ ID NO:182; B4701 and DRB 1 0102, SEQ ID NOs: 148 and 149; A3101, SEQ ID NO:145; B4402, SEQ ID NOs:158 and 166; B3503, SEQ ID NOs:146 and 162; DRB 1 1104, SEQ ID NO:149.
  • H2N refers to the N-terminal end of the peptide, and —COOH the C-terminus.

Abstract

This invention provides peptides, immunogenic compositions and vaccines, and methods of treating, reducing the incidence of, and inducing immune responses to a WT-1-expressing cancer, comprising peptides derived from the WT-1 protein.

Description

  • This application claims priority to U.S. provisional application Ser. Nos. 61/586,177, filed Jan. 13, 2012; and Ser. No. 61/647,207, filed May 15, 2012; both of which are incorporated herein by reference in their entireties.
    • GOVERNMENT SUPPORT
  • This work was supported by grants CA23766, CA59350 and CA08748 from the National Institutes of Health. The US government has certain rights in the invention.
    • FIELD OF INVENTION
  • This invention provides peptides, compositions and vaccines comprising same, and methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer, comprising administering same.
    • BACKGROUND OF THE INVENTION
  • Wilms tumor (WT), a pediatric nephroblastoma that occurs with a frequency of 1 in 10,000 births, has been the subject of intense clinical and basic research for several years. The tumor is embryonic in origin; it is detected in children usually during the first 5 years of life and can occur unilaterally or bilaterally. A WT arises when condensed metanephric mesenchymal cells of the developing kidney fail to properly differentiate. The implication of the Wilms tumor 1 (WT1) tumor suppressor gene in the etiology of WT illustrated the impact that genetic alterations can have on both development and tumorigenesis.
  • Wilms tumor protein I (WT1) is a zinc finger transcription factor expressed during normal ontogenesis such as in fetal kidney, testis and ovary. In adults, WT1 expression is limited to low levels on hematopoietic stem cells, myoepithelial progenitor cells, renal podocytes and some cells in testis and ovary. Recent demonstration that WT1 is over expressed in several types of leukemia suggested that WT1 would be an attractive target for immunotherapy for various cancers.
    • SUMMARY OF THE INVENTION
  • This invention provides peptides, compositions, and immunogenic compositions such as vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer, comprising administering immunogenic peptides.
  • In one embodiment, the present invention provides an isolated WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:1-160, 162-185, 190, 191 and 193. In one embodiment, the present invention provides an isolated HLA class I binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 190, 191 and 193. In one embodiment, the present invention provides an isolated HLA class II binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:149, 156, 173, 174 and 180.
  • In one embodiment, the present invention provides an isolated WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO: 1-160, 162-185, 190, 191 and 193, or a fragment of any of the foregoing. In one embodiment, the present invention provides an isolated HLA class I binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 190, 191 and 193. In one embodiment, the present invention provides an isolated HLA class II binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:149, 156, 173, 174 and 180.
  • In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) a peptide selected from SEQ ID NO:1-160, 162-185, 190, 191 and 193. In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class I binding peptide selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class II binding peptide selected from SEQ ID NO:149, 156, 173, 174 and 180.
  • In another embodiment, the present invention provides a vaccine comprising one or more peptides of SEQ ID NO:1-160, 162-185, 190, 191 and 193. In another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptides selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. In another embodiment, the present invention provides a vaccine comprising one or more HLA class II binding peptides selected from SEQ ID NO:149, 156, 173, 174 and 180. In another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptides selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, and one or more HLA class II binding peptides selected from SEQ ID NO:149, 156, 173, 174 and 180.
  • In another embodiment, the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • In another embodiment, the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.
  • In another embodiment, the present invention provides a method of inducing formation and proliferation of a WT1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of a WT1 protein-specific CTL. This method can be conducted in vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect.
  • In another embodiment, the present invention provides a method of inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein; or a combination thereof. This method can be conducted in vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect.
  • In another embodiment, the present invention provides a method of inducing an anti-cancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby inducing an anti-mesothelioma immune response in a subject. In one embodiment, the fragment of a WT1 protein consists of a peptide or comprises a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 and 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.
  • In another embodiment, the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby treating a subject with a mesothelioma. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.
  • In another embodiment, the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.
  • In another embodiment, the cancer is a WT1-expressing cancer. In one embodiment, the WT1-expressing cancer is an acute myelogenous leukemia (AML). In another embodiment, the WT1-expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT1-expressing cancer is an MDS. In another embodiment, the WT1-expressing cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the WT1-expressing cancer is a Wilms' tumor. In another embodiment, the WT1-expressing cancer is a leukemia. In another embodiment, the WT1-expressing cancer is a hematological cancer. In another embodiment, the WT1-expressing cancer is a lymphoma. In another embodiment, the WT1-expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT1-expressing cancer is a mesothelioma. In another embodiment, the WT1-expressing cancer is a malignant mesothelioma. In another embodiment, the WT1-expressing cancer is a gastric cancer. In another embodiment, the WT1-expressing cancer is a colon cancer. In another embodiment, the WT1-expressing cancer is a lung cancer. In another embodiment, the WT1-expressing cancer is abreast cancer. In another embodiment, the WT1-expressing cancer is a germ cell tumor. In another embodiment, the WT1-expressing cancer is an ovarian cancer. In another embodiment, the WT1-expressing cancer is a uterine cancer. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a liver cancer. In another embodiment, the WT1-expressing cancer is a renal cancer. In another embodiment, the WT1-expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT1-expressing cancer is a sarcoma. In another embodiment, the WT1-expressing cancer is any other carcinoma or sarcoma.
  • In another embodiment, the WT1-expressing cancer is a solid tumor. In another embodiment, the solid tumor is associated with a WT1-expressing cancer. In another embodiment, the solid tumor is associated with a myelodysplastic syndrome (MDS). In another embodiment, the solid tumor is associated with a non-small cell lung cancer (NSCLC). In another embodiment, the solid tumor is associated with a lung cancer. In another embodiment, the solid tumor is associated with a breast cancer. In another embodiment, the solid tumor is associated with a colorectal cancer. In another embodiment, the solid tumor is associated with a prostate cancer. In another embodiment, the solid tumor is associated with an ovarian cancer. In another embodiment, the solid tumor is associated with a renal cancer. In another embodiment, the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
  • In another embodiment, the present invention provides a composition comprising an isolated peptide of the invention in combination with at least 1 additional WT1 peptide. In certain embodiments, a composition comprising at least 2 different isolated peptides of the present invention is provided. In certain embodiments, a composition comprising at least 3 or at least 4 different isolated peptides of the present invention is provided. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the composition of the present invention is a vaccine.
  • In another embodiment, the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a peptide or composition of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • In another embodiment, the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a peptide or composition of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.
  • In another embodiment, the present invention provides a method of inducing formation and proliferation of a WT1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of a WT1 protein-specific CTL.
  • In another embodiment, the present invention provides a method of inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein; or a combination thereof.
  • In another embodiment, the invention is directed to a peptide of the invention with at least one amino acid change that increases the affinity of the peptide for binding to a HLA molecule.
    • BRIEF DESCRIPTION OF THE FIGURES
  • So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention are briefly summarized. Details of the above may be had by reference to certain embodiments thereof, which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted; however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.
  • FIG. 1 A-D shows WT1 specific responses of CTL generated from PBMC of normal donors (n=56) by stimulation with autologous APCs loaded with total pool of WT1 derived pentadecapeptides;
  • FIG. 2 A-E depicts the strategy for the generation of the total pool of overlapping pentadecapeptides spanning the whole sequence of the WT1 protein and epitope mapping;
  • FIG. 3 A-D shows that the combined HLA class I and II restricted WT1 specific T cell response to the same immunodominant peptide sequence derived from WT1 protein in the WT1 CTL after 40 days of co-culture with the WT1 total pool of overlapping 15-mers loaded on autologous CAMs;
  • FIG. 4A-F depicts schema of WT1; and
  • FIG. 5 depicts results using mixed A0201 epitopes loaded on A0201-AAPC in 8 normal A0201+ donors.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • This invention provides immunogenic peptides, and compositions and vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer, comprising administering one or more immunogenic peptides.
  • This invention provides WT1 peptides and methods of treating, reducing the incidence of, and inducing immune responses against a WT1-expressing cancer, comprising immunogenic peptides.
  • The WT1 molecule from which the peptides of the present invention are derived has, in another embodiment, the sequence:
  • (SEQ ID NO: 194)
    1 SRQRPHPGAL RNPTACPLPH FPPSLPPTHS PTHPPRAGTA
    AQAPGPRRLL
    51 AAILDFLLLQ DPASTCVPEP ASQHTLRSGP GCLQQPEQQG
    VRDPGGIWAK
    101 LGAAEASAER LQGRRSRGAS GSEPQQMGSD VRDLNALLPA
    VPSLGGGGGC
    151 ALPVSGAAQW APVLDFAPPG ASAYGSLGGP APPPAPPPPP
    PPPPHSFIKQ
    201 EPSWGGAEPH EEQCLSAFTV HFSGQFTGTA GACRYGPFGP
    PPPSQASSGQ
    251 ARMFPNAPYL PSCLESQPAI RNQGYSTVTF DGTPSYGHTP
    SHHAAQFPNH
    301 SFKHEDPMGQ QGSLGEQQYS VPPPVYGCHT PTDSCTGSQA
    LLLRTPYSSD
    351 NLYQMTSQLE CMTWNQMNLG ATLKGVAAGS SSSVKWTEGQ
    SNHSTGYESD
    401 NHTTPILCGA QYRIHTHGVF RGIQDVRRVP GVAPTLVRSA
    SETSEKRPFM
    451 CAYPGCNKRY FKLSHLQMHS RKHTGEKPYQ CDFKDCERRF
    SRSDQLKRHQ
    501 RRHTGVKPFQ CKTCQRKFSR SDHLKTHTRT HTGKTSEKPF
    SCRWPSCQKK
    551 FARSDELVRH HNMHQRNMTK LQLAL

    The foregoing sequence of the WT1 protein is that published by Gessler et al. (37) which comprises 575 aminoacids and includes the first 126 aminoacids in the N-terminus missing in the (Exon 5+, KTS+) isoform of WT116.
  • In another embodiment, the WT1 sequence is
  • MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGS
    LGGPAPPPAPP
    PPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGP
    FGPPPPSQASSGQA
    RMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHS
    FKHEDPMGQQGS
    LGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMT
    WNQMNLGATLK
    GVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQ
    DVRRVPGVAPTL
    VRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDC
    ERRFSRSDQLK
    RHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSC
    QKKFARSDELVR HHNMHQRNMTKLQLAL (GenBank Accession
    number AY245105; SEQ ID NO: 195).
  • In another embodiment, the WT1 molecule has the sequence:
  • AAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCAL
    PVSGAAQWAP
    VLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEE
    QCLSAFTVHFSGQF
    TGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYS
    TVTFDGTPSYGHT
    PSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQ
    ALLLRTPYSSDN
    LYQMTSQLECMTWNQMNLGATLKGHSTGYESDNHTTPILCGAQYRIHTHG
    VFRGIQDVRRV
    PGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPY
    QCDFKDCERRF
    SRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCR
    WPSCQKKFARS DELVRHHNMHQRNMTKLQLAL (GenBank
    Accession number NM_000378; SEQ ID NO: 196).
  • In another embodiment, the WT1 molecule has the sequence:
  • MQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASA
    ERLQGRRSRGA
    SGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPP
    GASAYGSLGGP
    APPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTA
    GACRYGPFGPPPPSQ
    ASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAA
    QFPNHSFKHEDP
    MGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTS
    QLECMTWNQM
    NLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTH
    GVFRGIQDVRRV
    PGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPY
    QCDFKDCERRF
    SRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCR
    WPSCQKKFARS DELVRHHNMHQRNMTKLQLAL (GenBank
    Accession number NP_077742; SEQ ID No: 197).
  • In another embodiment, the WT1 protein has the sequence set forth in GenBank Accession # NM_024426. In other embodiments, the WT1 protein has or comprises one of the sequences set forth in one of the following sequence entries: NM_024425, NM_024424, NM_000378, 595530, D13624, D12496, D 12497, or X77549. In another embodiment, the WT1 protein has any other WT1 sequence known in the art. This invention provides peptides, compositions, and immunogenic compositions such as vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer, comprising administering immunogenic peptides.
  • In one embodiment, the present invention provides an isolated WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:1-160, 162-185, 190, 191 and 193. In one embodiment, the present invention provides an isolated HLA class I binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. In one embodiment, the present invention provides an isolated HLA class II binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:149, 156, 173, 174 and 180. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO:149.
  • In one embodiment, the present invention provides an isolated WT1 peptide having an amino acid (AA) sequence comprising any one of the sequences SEQ ID NO:1-53 or 43-XXX, or a fragment thereof. In one embodiment, the present invention provides an isolated HLA class I binding WT1 peptide having an amino acid (AA) sequence comprising of any one of the sequences SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. In one embodiment, the present invention provides an isolated HLA class II binding WT1 peptide having an amino acid (AA) sequence comprising of any one of the sequences SEQ ID NO:149, 156, 173, 174 and 180. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO:149.
  • In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) a peptide selected from SEQ ID NO:1-160, 162-185, 190, 191 and 193. In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class I binding peptide selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class II binding peptide selected from SEQ ID NO:149, 156, 173, 174 and 180. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO:149.
  • In another embodiment, the present invention provides a vaccine comprising one or more peptides of SEQ ID NO:1-160, 162-185, 190, 191 and 193. In another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptides selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183. In another embodiment, the present invention provides a vaccine comprising one or more HLA class II binding peptides selected from SEQ ID NO:149, 156, 173, 174 and 180. In another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptides selected from SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, and one or more HLA class II binding peptides selected from SEQ ID NO:149, 156, 173, 174 and 180. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO:149.
  • In another embodiment, the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • In another embodiment, the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.
  • In another embodiment, the present invention provides a method of inducing an anti-cancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby inducing an anti-mesothelioma immune response in a subject. In one embodiment, the fragment of a WT1 protein consists of a peptide or comprises a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 and 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180.
  • In another embodiment, the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby treating a subject with a mesothelioma. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 and 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO:149.
  • In another embodiment, the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO:149.
  • In another embodiment, the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • In another embodiment, the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.
  • In another embodiment, the present invention provides a method of inducing an anti-cancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby inducing an anti-mesothelioma immune response in a subject. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, or SEQ ID NO:149, 156, 173, 174 and 180. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO:149.
  • In another embodiment, the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby treating a subject with a mesothelioma. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193.
  • In another embodiment, the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:1-160, 162-185, 190, 191 or 193.
  • In another embodiment, the cancer is a WT1-expressing cancer. In one embodiment, the WT1-expressing cancer is an acute myelogenous leukemia (AML). In another embodiment, the WT1-expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT1-expressing cancer is an MDS. In another embodiment, the WT1-expressing cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the WT1-expressing cancer is a Wilms' tumor. In another embodiment, the WT1-expressing cancer is a leukemia. In another embodiment, the WT1-expressing cancer is a hematological cancer. In another embodiment, the WT1-expressing cancer is a lymphoma. In another embodiment, the WT1-expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT1-expressing cancer is a mesothelioma. In another embodiment, the WT1-expressing cancer is a malignant mesothelioma. In another embodiment, the WT1-expressing cancer is a gastric cancer. In another embodiment, the WT1-expressing cancer is a colon cancer. In another embodiment, the WT1-expressing cancer is a lung cancer. In another embodiment, the WT1-expressing cancer is abreast cancer. In another embodiment, the WT1-expressing cancer is a germ cell tumor. In another embodiment, the WT1-expressing cancer is an ovarian cancer. In another embodiment, the WT1-expressing cancer is a uterine cancer. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a liver cancer. In another embodiment, the WT1-expressing cancer is a renal cancer. In another embodiment, the WT1-expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT1-expressing cancer is a sarcoma. In another embodiment, the WT1-expressing cancer is any other carcinoma or sarcoma.
  • In another embodiment, the WT1-expressing cancer is a solid tumor. In another embodiment, the solid tumor is associated with a WT1-expressing cancer. In another embodiment, the solid tumor is associated with a myelodysplastic syndrome (MDS). In another embodiment, the solid tumor is associated with a non-small cell lung cancer (NSCLC). In another embodiment, the solid tumor is associated with a lung cancer. In another embodiment, the solid tumor is associated with a breast cancer. In another embodiment, the solid tumor is associated with a colorectal cancer. In another embodiment, the solid tumor is associated with a prostate cancer. In another embodiment, the solid tumor is associated with an ovarian cancer. In another embodiment, the solid tumor is associated with a renal cancer. In another embodiment, the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
  • In another embodiment, the present invention provides a composition comprising an isolated peptide of the invention in combination with at least 1 additional WT1 peptide. In certain embodiments, a composition comprising at least 2 different isolated peptides of the present invention is provided. In certain embodiments, a composition comprising at least 3 or at least 4 different isolated peptides of the present invention is provided. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the composition of the present invention is a vaccine.
  • In another embodiment, the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a peptide or composition of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • In another embodiment, the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a peptide or composition of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject.
  • In another embodiment, the present invention provides a method of inducing formation and proliferation of a WT1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of a WT1 protein-specific CTL.
  • In another embodiment, the present invention provides a method of inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein; or a combination thereof.
  • In another embodiment, the invention is directed to a peptide of the invention with at least one amino acid change that increases the affinity of the peptide for binding to a HLA molecule.
  • “Peptide,” in another embodiment of methods and compositions of the present invention, refers to a compound of subunit AA connected by peptide bonds. In another embodiment, the peptide comprises an AA analogue. In another embodiment, the peptide comprises a peptidomimetic. The different AA analogues and peptidomimetics that can be included in the peptides of methods and compositions of the present invention are enumerated herein below. The subunits are, in another embodiment, linked by peptide bonds. In another embodiment, the subunit is linked by another type of bond, e.g. ester, ether, etc. Each possibility represents a separate embodiment of the present invention.
  • The unaltered peptides of the present invention (as described both above and below) are referred to collectively herein as “WT1 peptides.” Each of the embodiments enumerated below for “WT1 peptides” applies to unaltered WT1 peptides and HLA class I and class II heteroclitic peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, a WT1 peptide of the present invention binds to an HLA class I molecule or a class II molecule. In another embodiment the peptide binds to both a class I and a class II molecule. In another embodiment, the HLA class II molecule is an HLA-DRB molecule. In another embodiment, the HLA class Il-molecule is an HLA-DRA molecule. In another embodiment, the HLA molecule is an HLA-DQA1 molecule. In another embodiment, the HLA molecule is an HLA-DQB1 molecule. In another embodiment, the HLA molecule is an HLA-DPA1 molecule. In another embodiment, the HLA molecule is an HLA-DPB 1 molecule. In another embodiment, the HLA molecule is an HLA-DMA molecule. In another embodiment, the HLA molecule is an HLA-DMB molecule. In another embodiment, the HLA molecule is an HLA-DOA molecule. In another embodiment, the HLA molecule is an HLA-DOB molecule. In another embodiment, the HLA molecule is any other HLA class Il-molecule known in the art. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the HLA class I molecule whose binding motif is contained in or comprising a peptide of the present invention is, in another embodiment, an HLA-A molecule. In another embodiment, the HLA class I molecule is an HLA-B molecule. In another embodiment, the HLA class I molecule is an HLA-C molecule. In another embodiment, the HLA class I molecule is an HLA-A0201 molecule. In another embodiment, the molecule is HLA A1. In another embodiment, the HLA class I molecule is HLA A2. In another embodiment, the HLA class I molecule is HLA A2.1. In another embodiment, the HLA class I molecule is HLA A3. In another embodiment, the HLA class I molecule is HLA A3.2. In another embodiment, the HLA class I molecule is HLA A11. In another embodiment, the HLA class I molecule is HLA A24. In another embodiment, the HLA class I molecule is HLA B7. In another embodiment, the HLA class I molecule is HLA B27. In another embodiment, the HLA class I molecule is HLA B8. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the HLA class I molecule-binding WT1 peptide of methods and compositions of the present invention binds to a superfamily of HLA class I molecules. In another embodiment, the superfamily is the A2 superfamily. In another embodiment, the superfamily is the A3 superfamily. In another embodiment, the superfamily is the A24 superfamily. In another embodiment, the superfamily is the B7 superfamily. In another embodiment, the superfamily is the B27 superfamily. In another embodiment, the superfamily is the B44 superfamily. In another embodiment, the superfamily is the C1 superfamily. In another embodiment, the superfamily is the C4 superfamily. In another embodiment, the superfamily is any other superfamily known in the art. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the HLA molecule is a A0101, A0201, A0203, A2402, A6901, B0702, A3101, B3501, B3503, B3508, B3802, B3801, B3901, B4001, B4402, B4701, B5701, C0401, C1701, DRB 10101, DRB 10402, DRB 10402, DRB10401 or DRB 11104 molecule. In another embodiment, the peptides of SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, and 183, and SEQ ID NO:149, 156, 173, 174 and 180, bind to the HLA class I or class II molecules described for each peptide in Tables 1 or 2. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:142, 143, 144, 145, 146, 147, 148, 150 or 151, and the HLA class II peptide consists of or comprises SEQ ID NO:149, and bind to the corresponding HLA molecule or molecules indicated for each peptide in Table 1 or Table 2. In one embodiment, certain peptides can bind to more than one HLA allele.
  • In another embodiment, a modification of a peptide of the invention is provided. In one embodiment the modification comprises at least one heteroclitic amino acid change, also referred to as a mutation or mutated, or an anchor residue mutation (see below). An HLA class I molecule binding motif of a modified peptide of the present invention exhibits an increased affinity for the HLA class I molecule, relative to the unmutated counterpart of the peptide. In another embodiment, the point mutation increases the affinity of the isolated, mutated WT1 peptide for the HLA class I molecule. In another embodiment, the increase in affinity is relative to the affinity (for the same HLA class I molecule) of the isolated, unmutated WT1 peptide wherefrom the isolated, mutated WT1 peptide was derived. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, a WT1 peptide of methods and compositions of the present invention is so designed as to exhibit affinity for an HLA molecule. In another embodiment, the affinity is a high affinity, as described herein.
  • HLA molecules, known in another embodiment as major histocompatibility complex (MHC) molecules, bind peptides and present them to immune cells. Thus, in another embodiment, the immunogenicity of a peptide is partially determined by its affinity for HLA molecules. HLA class I molecules interact with CD8 molecules, which are generally present on cytotoxic T lymphocytes (CTL). HLA class II molecules interact with CD4 molecules, which are generally present on helper T lymphocytes.
  • In another embodiment, a peptide of the present invention is immunogenic. In another embodiment, “immunogenic” refers to an ability to stimulate, elicit or participate in an immune response. In another embodiment, the immune response elicited is a cell-mediated immune response. In another embodiment, the immune response is a combination of cell-mediated and humoral responses.
  • In another embodiment, T cells that bind to the MHC molecule-peptide complex become activated and induced to proliferate and lyse cells expressing a protein comprising the peptide. T cells are typically initially activated by “professional” antigen presenting cells (“APC”; e.g. dendritic cells, monocytes, and macrophages), which present costimulatory molecules that encourage T cell activation as opposed to anergy or apoptosis. In another embodiment, the response is heteroclitic, as described herein, such that the CTL lyses a neoplastic cell expressing a protein which has an AA sequence homologous to a peptide of this invention, or a different peptide than that used to first stimulate the T cell.
  • In another embodiment, an encounter of a T cell with a peptide of this invention induces its differentiation into an effector and/or memory T cell. Subsequent encounters between the effector or memory T cell and the same peptide, or, in another embodiment, with a related peptide of this invention, leads to a faster and more intense immune response. Such responses are gauged, in another embodiment, by measuring the degree of proliferation of the T cell population exposed to the peptide. In another embodiment, such responses are gauged by any of the methods enumerated herein below.
  • In another embodiment, the peptides of methods and compositions of the present invention bind an HLA class II molecule with high affinity. In other embodiments, the HLA class II molecule is any HLA class II molecule enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, derivatives of peptides of methods and compositions of the present invention bind an HLA class I molecule with high affinity. In other embodiments, the MHC class I molecule is any MHC class I molecule enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, a peptide of methods and compositions of the present invention binds an HLA class II molecule with significant affinity, while a peptide derived from the original peptide binds an HLA class I molecule with significant affinity.
  • In another embodiment, “affinity” refers to the concentration of peptide necessary for inhibiting binding of a standard peptide to the indicated MHC molecule by 50%. In another embodiment, “high affinity” refers to an affinity is such that a concentration of about 500 nanomolar (nM) or less of the peptide is required for 50% inhibition of binding of a standard peptide. In another embodiment, a concentration of about 400 nM or less of the peptide is required. In another embodiment, the binding affinity is 300 nM. In another embodiment, the binding affinity is 200 nM. In another embodiment, the binding affinity is 150 nM. In another embodiment, the binding affinity is 100 nM. In another embodiment, the binding affinity is 80 nM. In another embodiment, the binding affinity is 60 nM. In another embodiment, the binding affinity is 40 nM. In another embodiment, the binding affinity is 30 nM. In another embodiment, the binding affinity is 20 nM. In another embodiment, the binding affinity is 15 nM. In another embodiment, the binding affinity is 10 nM. In another embodiment, the binding affinity is 8 nM. In another embodiment, the binding affinity is 6 nM. In another embodiment, the binding affinity is 4 nM. In another embodiment, the binding affinity is 3 nM. In another embodiment, the binding affinity is 2 nM. In another embodiment, the binding affinity is 1.5 nM. In another embodiment, the binding affinity is 1 nM. In another embodiment, the binding affinity is 0.8 nM. In another embodiment, the binding affinity is 0.6 nM. In another embodiment, the binding affinity is 0.5 nM. In another embodiment, the binding affinity is 0.4 nM. In another embodiment, the binding affinity is 0.3 nM. In another embodiment, the binding affinity is less than 0.3 nM.
  • In another embodiment, “affinity” refers to a measure of binding strength to the MHC molecule. In another embodiment, affinity is measured using a method known in the art to measure competitive binding affinities. In another embodiment, affinity is measured using a method known in the art to measure relative binding affinities. In another embodiment, the method is a competitive binding assay. In another embodiment, the method is radioimmunoassay or RIA. In another embodiment, the method is BiaCore analyses. In another embodiment, the method is any other method known in the art. In another embodiment, the method yields an IC50 in relation to an IC50 of a reference peptide of known affinity.
  • Each type of affinity and method of measuring affinity represents a separate embodiment of the present invention.
  • In another embodiment, “high affinity” refers to an IC50 of 0.5-500 nM. In another embodiment, the IC50 is 1-300 nM. In another embodiment, the IC50 is 1.5-200 nM. In another embodiment, the IC50 is 2-100 nM. In another embodiment, the IC50 is 3-100 nM. In another embodiment, the IC50 is 4-100 nM. In another embodiment, the IC50 is 6-100 nM. In another embodiment, the IC50 is 10-100 nM. In another embodiment, the IC50 is 30-100 nM. In another embodiment, the IC50 is 3-80 nM. In another embodiment, the IC50 is 4-60 nM. In another embodiment, the IC50 is 5-50 nM. In another embodiment, the IC50 is 6-50 nM. In another embodiment, the IC50 is 8-50 nM. In another embodiment, the IC50 is 10-50 nM. In another embodiment, the IC50 is 20-50 nM. In another embodiment, the IC50 is 6-40 nM. In another embodiment, the IC50 is 8-30 nM. In another embodiment, the IC50 is 10-25 nM. In another embodiment, the IC50 is 15-25 nM. Each affinity and range of affinities represents a separate embodiment of the present invention.
  • In another embodiment, a peptide of methods and compositions of the present invention binds to a superfamily of HLA molecules. Superfamilies of HLA molecules share very similar or identical binding motifs. In another embodiment, the superfamily is a HLA class I superfamily. In another embodiment, the superfamily is a HLA class II superfamily. Each possibility represents a separate embodiment of the present invention.
  • The terms “HLA-binding peptide,” “HLA class I molecule-binding peptide,” and “HLA class II molecule-binding peptide” refer, in another embodiment, to a peptide that binds an HLA molecule with measurable affinity. In another embodiment, the terms refer to a peptide that binds an HLA molecule with high affinity. In another embodiment, the terms refer to a peptide that binds an HLA molecule with sufficient affinity to activate a T cell precursor. In another embodiment, the terms refer to a peptide that binds an HLA molecule with sufficient affinity to mediate recognition by a T cell. The HLA molecule is, in other embodiments, any of the HLA molecules enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • “Heteroclitic” refers, in another embodiment, to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived (e.g. the peptide not containing the anchor residue mutations). In another embodiment, “original peptide” refers to a peptide of the present invention. In another embodiment, “heteroclitic” refers to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide. In another embodiment, a “heteroclitic” immune response refers to an immune response that recognizes the original peptide from which the improved peptide was derived (e.g. the peptide not containing the anchor residue mutations). In another embodiment, a “heteroclitic” immune response refers to an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide. In another embodiment, the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response substantially equal to the response to vaccination with the original peptide. In another embodiment, the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response less than the response to vaccination with the original peptide. In another embodiment, a heteroclitic peptide of the present invention is an HLA class I heteroclitic peptide. Methods for identifying HLA class I and class II residues, and for improving HLA binding by mutating the residues, are well known in the art, as described below. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, a heteroclitic peptide of the present invention induces an immune response that is increased at least 2-fold relative to the WT1 peptide from which the heteroclitic peptide was derived (“native peptide”). In another embodiment, the increase is 3-fold relative to the native peptide. In another embodiment, the increase is 5-fold relative to the native peptide. In another embodiment, the increase is 7-fold relative to the native peptide. In another embodiment, the increase is 10-fold relative to the native peptide. In another embodiment, the increase is 15-fold relative to the native peptide. In another embodiment, the increase is 20-fold relative to the native peptide. In another embodiment, the increase is 30-fold relative to the native peptide. In another embodiment, the increase is 50-fold relative to the native peptide. In another embodiment, the increase is 100-fold relative to the native peptide. In another embodiment, the increase is 150-fold relative to the native peptide. In another embodiment, the increase is 200-fold relative to the native peptide. In another embodiment, the increase is 300-fold relative to the native peptide. In another embodiment, the increase is 500-fold relative to the native peptide. In another embodiment, the increase is 1000-fold relative to the native peptide. In another embodiment, the increase is more than 1000-fold relative to the native peptide. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the present invention provides a HLA class II heteroclitic peptide derived from an isolated WT1 peptide of the present invention. In another embodiment, the process of deriving comprises introducing a mutation that enhances a binding of the peptide to an HLA class II molecule. In another embodiment, the process of deriving consists of introducing a mutation that enhances a binding of the peptide to an HLA class I molecule. In another embodiment, the mutation is in an HLA class II anchor residue. In another embodiment, a heteroclitic class II peptide of the present invention is identified and tested in a manner analogous to identification and testing of HLA class I heteroclitic peptides, as exemplified herein. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the HLA class II binding site in a peptide of the present invention is created or improved by mutation of an HLA class II motif anchor residue. In another embodiment, the anchor residue that is modified is in the P1 position. In another embodiment, the anchor residue is at the P2 position. In another embodiment, the anchor residue is at the P6 position. In another embodiment, the anchor residue is at the P9 position. In another embodiment, the anchor residue is selected from the P1, P2, P6, and P9 positions. In another embodiment, the anchor residue is at the P3 position. In another embodiment, the anchor residue is at the P4 position. In another embodiment, the anchor residue is at the P5 position. In another embodiment, the anchor residue is at the P6 position. In another embodiment, the anchor residue is at the P8 position. In another embodiment, the anchor residue is at the P10 position. In another embodiment, the anchor residue is at the P11 position. In another embodiment, the anchor residue is at the P12 position. In another embodiment, the anchor residue is at the P13 position. In another embodiment, the anchor residue is at any other anchor residue of an HLA class II molecule that is known in the art. In another embodiment, residues other than P1, P2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve HLA class II binding. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, a heteroclitic peptide is generated by introduction of a mutation that creates an anchor motif. “Anchor motifs” or “anchor residues” refers, in another embodiment, to 1 or a set of preferred residues at particular positions in an HLA-binding sequence.
  • HLA-binding sequence is an HLA class II-binding sequence. In another embodiment, the HLA-binding sequence is an HLA class I-binding sequence. In another embodiment, the positions corresponding to the anchor motifs are those that play a significant role in binding the HLA molecule. In another embodiment, the anchor residue is a primary anchor motif. In another embodiment, the anchor residue is a secondary anchor motif. Each possibility represents a separate embodiment of the present invention.
  • Methods for predicting MHC class H epitopes are well known in the art. In another embodiment, the MHC class II epitope is predicted using TEPITOPE (Meister G E, Roberts C G et al, Vaccine 1995 13: 581-91). In another embodiment, the MHC class II epitope is predicted using EpiMatrix (De Groot A S, Jesdale B M et al, AIDS Res Hum Retroviruses 1997 13: 529-31). In another embodiment, the MHC class II epitope is predicted using the Predict Method (Yu K, Petrovsky N et al, Mol Med. 2002 8: 137-48). In another embodiment, the MHC class II epitope is predicted using the SYFPEITHI epitope prediction algorithm (Examples). In another embodiment, the MHC class II epitope is predicted using Rankpep. In another embodiment, the MHC class II epitope is predicted using any other method known in the art. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, in the case of HLA class II-binding peptides (e.g. HLA-DR-binding peptides), the anchor residue that is modified is in the P1 position. In another embodiment, the anchor residue is in the P2 position. In another embodiment, the anchor residue is in the P6 position. In another embodiment, the anchor residue is in the P9 position. In other embodiments, the anchor residue is the P3, P4, P5, P6, P8, P10, P11, P12, or P13 position. In another embodiment, the anchor residue is any other anchor residue of an HLA class II molecule that is known in the art. In another embodiment, residues other than P1, P2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve HLA class II binding. In another embodiment, any combination of the above residues is mutated. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, a WT1 peptide of the present invention binds to 2 distinct HLA class II molecules. In another embodiment, the peptide binds to three distinct HLA class II molecules. In another embodiment, the peptide binds to four distinct HLA class II molecules. In another embodiment, the peptide binds to five distinct HLA class II molecules. In another embodiment, the peptide binds to six distinct HLA class II molecules. In another embodiment, the peptide binds to more than six distinct HLA class II molecules.
  • In another embodiment, the HLA class II molecules that are bound by a WT1 peptide of the present invention are encoded by two or more distinct alleles at a given HLA class II locus. In another embodiment, the HLA class II molecules are encoded by 3 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 4 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 5 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 6 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by more than six distinct alleles at a locus.
  • In another embodiment, the HLA class II molecules bound by the WT1 peptide are encoded by HLA class II genes at 2 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 2 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 3 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 3 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 4 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 4 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at more than 4 distinct loci. In other embodiments, the loci are selected from HLA-DRB loci. In another embodiment, the HLA class II-binding peptide is an HLA-DRA binding peptide. In another embodiment, the peptide is an HLA-DQA1 binding peptide. In another embodiment, the peptide is an HLA-DQB 1 binding peptide. In another embodiment, the peptide is an HLA-DPA1 binding peptide. In another embodiment, the peptide is an HLA-DPB 1 binding peptide. In another embodiment, the peptide is an HLA-DMA binding peptide. In another embodiment, the peptide is an HLA-DMB binding peptide. In another embodiment, the peptide is an HLA-DOA binding peptide. In another embodiment, the peptide is an HLA-DOB binding peptide. In another embodiment, the peptide binds to any other HLA class II molecule known in the art. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, a WT1 peptide of the present invention binds to 2 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 3 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 4 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 5 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 6 distinct HLA-DRB molecules. In another embodiment, the peptide binds to more than 6 distinct HLA-DRB molecules.
  • In another embodiment, a WT1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 3 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 4 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 5 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 6 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by more than 6 distinct HLA-DRB alleles. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, a WT1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by 3 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by 4 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by 5 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, DRB 1104 and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by each of the following HLA-DRB alleles: DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the present invention provides a composition comprising 2 distinct WT1 peptides of the present invention. In another embodiment, the 2 distinct WT1 peptides are both unaltered. In another embodiment, 1 of the WT1 peptides is unaltered, while the other is heteroclitic. In another embodiment, both of the WT1 peptides are heteroclitic.
  • In another embodiment, the composition comprises 3 distinct WT1 peptides of the present invention. In another embodiment, the composition comprises 4 distinct WT1 peptides of the present invention. In another embodiment, the composition comprises 5 distinct WT1 peptides of the present invention. In another embodiment, the composition comprises more than 5 distinct isolated WT1 peptides of the present invention.
  • In another embodiment, 2 of the WT1 peptides in the composition are unaltered. In another embodiment, 2 of the WT1 peptides in the composition are heteroclitic. In another embodiment, 2 of the WT1 peptides in the composition are unaltered, and 2 are heteroclitic. In another embodiment, more than 2 of the WT1 peptides in the composition are unaltered. In another embodiment, more than 2 of the WT1 peptides in the composition are heteroclitic. In another embodiment, more than 2 of the WT1 peptides in the composition are unaltered, and more than 2 are heteroclitic. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, 1 of the additional WT1 peptides in a composition of the present invention has a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193. In another embodiment, 2 of the additional WT1 peptides have a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193. In another embodiment, 3 of the additional WT1 peptides have a sequence selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193.
  • In another embodiment, any other immunogenic WT1 peptide known in the art is utilized as an additional WT1 peptide. In another embodiment, any combination of immunogenic WT1 peptides known in the art is utilized. Non-limiting sources of other WT peptides include WO2005053618, WO2007047764 and WO2007120673.
  • Each additional WT1 peptide, and each combination thereof, represents a separate embodiment of the present invention.
  • In another embodiment, a composition of the present invention contains 2 HLA class II heteroclitic peptides that are derived from the same isolated WT1 peptide of the present invention. In another embodiment, the 2 HLA class II heteroclitic peptides contain mutations in different HLA class II molecule anchor residues. In another embodiment, the 2 HLA class II heteroclitic peptides contain different mutations in the same anchor residues. In another embodiment, 2 of the HLA class II heteroclitic peptides are derived from different isolated WT1 peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, 2 WT1 peptides of the present invention, or the WT1 peptides that correspond to two HLA class II heteroclitic peptides of the present invention, overlap with one another. In another embodiment, the overlap between the peptides is at least 7 amino acids (AA). In another embodiment, the overlap is at least 8 AA. In another embodiment, the overlap is at least 9 AA. In another embodiment, the overlap is 7 AA. In another embodiment, the overlap is 8 AA. In another embodiment, the overlap is 9 AA. In another embodiment, the overlap is 10 AA. In another embodiment, the overlap is 11 AA. In another embodiment, the overlap is 12 AA. In another embodiment, the overlap is 13 AA. In another embodiment, the overlap is 14 AA. In another embodiment, the overlap is 15 AA. In another embodiment, the overlap is 16 AA. In another embodiment, the overlap is more than 16 AA. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the peptides in a composition of the present invention bind to 2 distinct HLA class II molecules. In another embodiment, the peptides bind to 3 distinct HLA class II molecules. In another embodiment, the peptides bind to 4 distinct HLA class II molecules. In another embodiment, the peptides bind to 5 distinct HLA class II molecules. In another embodiment, the peptides bind to more than 5 distinct HLA class II molecules. In another embodiment, the peptides in the composition bind to the same HLA class II molecules.
  • In another embodiment, each of the WT 1 peptides in a composition of the present invention binds to a set of HLA class II molecules. In another embodiment, each of the WT1 peptides binds to a distinct set of HLA class II molecules. In another embodiment, the WT1 peptides in the composition bind to the same set of HLA class II molecules. In another embodiment, 2 of the WT1 peptides bind to a distinct but overlapping set of HLA class II molecules. In another embodiment, 2 or more of the WT1 peptides bind to the same set of HLA class II molecules, while another of the WT1 peptides binds to a distinct set. In another embodiment, 2 or more of the WT1 peptides bind to an overlapping set of HLA class II molecules, while another of the WT1 peptides binds to a distinct set.
  • In another embodiment, 2 or more of the WT1 peptides in a composition of the present invention each binds to more than 1 HLA-DRB molecule. In another embodiment, the 4 or more HLA-DRB molecules bound by the peptides in the composition are distinct from one another. In another embodiment, the HLA-DRB molecules are encoded by different HLA-DRB alleles. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, 2 or more of the HLA class II molecules bound by WT1 peptides in a composition of the present invention are HLA-DRB molecules. In another embodiment, 3 or more of the HLA class II molecules that are bound are HLA-DRB molecules. In other embodiments, the HLA class II molecules that are bound can be any of the HLA class II molecules enumerated herein. In another embodiment, the HLA class II molecules that are bound are encoded by 2 or more distinct HLA class II alleles at a given locus. In another embodiment, the HLA class II molecules that are bound are encoded by HLA class II genes at 2 or more distinct loci.
  • Each of the above compositions represents a separate embodiment of the present invention.
  • In another embodiment, a “set of HLA class II molecules” refers to the HLA class II molecules encoded by different alleles at a particular locus. In another embodiment, the term refers to HLA class II molecules with a particular binding specificity. In another embodiment, the term refers to HLA class II molecules with a particular peptide consensus sequence. In another embodiment, the term refers to a superfamily of HLA class II molecules. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the present invention provides a composition comprising an unaltered HLA class II molecule-binding WT1 peptide of the present invention and a second, HLA class I molecule-binding WT1 peptide. In another embodiment, the composition comprises more than 1 HLA class II molecule-binding WT1 peptide of the present invention, in addition to the HLA class I molecule-binding WT1 peptide. In another embodiment, the composition comprises more than 1 HLA class I molecule-binding WT1 peptide, in addition to the HLA class II molecule-binding WT1 peptide. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the AA sequence of the HLA class I molecule-binding WT1 peptide comprises a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193. In another embodiment, the AA sequence of the HLA class I molecule-binding WT1 peptide is selected from the sequences set forth in SEQ ID No: 1-160, 162-185, 190, 191 or 193. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the HLA class I molecule-binding WT1 peptide is an HLA class I heteroclitic peptide. In another embodiment, the HLA class I molecule-binding WT1 peptide contains a mutation in an HLA class I molecule anchor residue thereof, as described further herein. As provided herein, WT1-derived peptides were modified in HLA anchor residues to generate heteroclitic peptides with increased predicted binding to HLA-A0201 and HLA-A0301. Peptides with increased predicted binding also exhibited enhanced ability to bind HLA class I molecules and increased immunogenicity.
  • In another embodiment, the mutation that enhances MHC binding is in the residue at position 1 of the HLA class I heteroclitic peptide. In another embodiment, the residue is changed to tyrosine. In another embodiment, the residue is changed to glycine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to phenylalanine. In another embodiment, the residue is changed to any other residue known in the art. In another embodiment, a substitution in position 1 (e.g. to tyrosine) stabilizes the binding of the position 2 anchor residue.
  • In another embodiment, the mutation is in position 2 of the HLA class I heteroclitic peptide. In another embodiment, the residue is changed to leucine. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to isoleucine. In another embodiment, the residue is changed to methionine. In another embodiment, the residue is changed to any other residue known in the art.
  • In another embodiment, the mutation is in position 6 of the HLA class I heteroclitic peptide. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to cysteine. In another embodiment, the residue is changed to glutamine. In another embodiment, the residue is changed to histidine. In another embodiment, the residue is changed to any other residue known in the art.
  • In another embodiment, the mutation is in position 9 of the HLA class I heteroclitic peptide. In another embodiment, the mutation changes the residue at the C-terminal position thereof. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to isoleucine. In another embodiment, the residue is changed to leucine. In another embodiment, the residue is changed to alanine. In another embodiment, the residue is changed to cysteine. In another embodiment, the residue is changed to any other residue known in the art.
  • In another embodiment, the point mutation is in a primary anchor residue. In another embodiment, the HLA class I primary anchor residues are positions 2 and 9. In another embodiment, the point mutation is in a secondary anchor residue. In another embodiment, the HLA class I secondary anchor residues are positions 1 and 8. In another embodiment, the HLA class I secondary anchor residues are positions 1, 3, 6, 7, and 8. In another embodiment, the point mutation is in a position selected from positions 4, 5, and 8. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 2, 8, and 9 of the HLA class I binding motif. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 3, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 2, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 2, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 3, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 2 and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 6 and 9. Each possibility represents a separate embodiment of the present invention.
  • Each of the above anchor residues and substitutions represents a separate embodiment of the present invention.
  • In another embodiment, the HLA class I molecule-binding WT peptide has length of 9 AA. In another embodiment, the peptide has length of 10 AA. As provided herein, native and heteroclitic peptides of 9-10 AA exhibited substantial binding to HLA class I molecules and ability to elicit cytokine secretion and cytolysis by CTL.
  • In another embodiment, the HLA class I molecule that is bound by the HLA class I molecule-binding WT1 peptide is an HLA-A molecule. In another embodiment, the HLA class I-molecule is an HLA-A2 molecule. In another embodiment, the HLA class I-molecule is an HLA-A3 molecule. In another embodiment, the HLA class I-molecule is an HLA-A11 molecule. In another embodiment, the HLA class I-molecule is an HLA-B 8 molecule. In another embodiment, the HLA class I-molecule is an HLA-0201 molecule. In another embodiment, the HLA class I-molecule binds any other HLA class I molecule known in the art. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, a WT1 peptide of methods and compositions of the present invention has a length of 8-30 amino acids. In another embodiment, the peptide has a length of 9-11 AA. In another embodiment, the peptide ranges in size from 7-25 AA, or in another embodiment, 8-11, or in another embodiment, 8-15, or in another embodiment, 9-20, or in another embodiment, 9-18, or in another embodiment, 9-15, or in another embodiment, 8-12, or in another embodiment, 9-11 AA in length. In another embodiment, the peptide is 8 AA in length, or in another embodiment, 9 AA or in another embodiment, 10 AA or in another embodiment, 12 AA or in another embodiment, 25 AA in length, or in another embodiment, any length therebetween. In another embodiment, the peptide is of greater length, for example 50, or 100, or more. In this embodiment, the cell processes the peptide to a length of 7 and 25 AA in length. In this embodiment, the cell processes the peptide to a length of 9-11 AA Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the peptide is 15-23 AA in length. In another embodiment, the length is 15-24 AA. In another embodiment, the length is 15-25 AA. In another embodiment, the length is 15-26 AA. In another embodiment, the length is 15-27 AA. In another embodiment, the length is 15-28 AA. In another embodiment, the length is 14-30 AA. In another embodiment, the length is 14-29 AA. In another embodiment, the length is 14-28 AA. In another embodiment, the length is 14-26 AA. In another embodiment, the length is 14-24 AA. In another embodiment, the length is 14-22 AA. In another embodiment, the length is 14-20 AA. In another embodiment, the length is 16-30 AA. In another embodiment, the length is 16-28 AA. In another embodiment, the length is 16-26 AA. In another embodiment, the length is 16-24 AA. In another embodiment, the length is 16-22 AA. In another embodiment, the length is 18-30 AA. In another embodiment, the length is 18-28 AA. In another embodiment, the length is 18-26 AA. In another embodiment, the length is 18-24 AA. In another embodiment, the length is 18-22 AA. In another embodiment, the length is 18-20 AA. In another embodiment, the length is 20-30 AA. In another embodiment, the length is 20-28 AA. In another embodiment, the length is 20-26 AA. In another embodiment, the length is 20-24 AA. In another embodiment, the length is 22-30 AA. In another embodiment, the length is 22-28 AA. In another embodiment, the length is 22-26 AA. In another embodiment, the length is 24-30 AA. In another embodiment, the length is 24-28 AA. In another embodiment, the length is 24-26 AA.
  • Each of the above peptides, peptide lengths, and types of peptides represents a separate embodiment of the present invention.
  • In another embodiment, minor modifications are made to peptides of the present invention without decreasing their affinity for HLA molecules or changing their TCR specificity, utilizing principles well known in the art. In the case of HLA class I-binding peptides, “minor modifications” refers, in another embodiment, to e.g. insertion, deletion, or substitution of one AA, inclusive, or deletion or addition of 1-3 AA outside of the residues between 2 and 9, inclusive. While the computer algorithms described herein are useful for predicting the MHC class I-binding potential of peptides, they have 60-80% predictive accuracy; and thus, the peptides should be evaluated empirically before a final determination of MHC class I-binding affinity is made. Thus, peptides of the present invention are not limited to peptides predicated by the algorithms to exhibit strong MHC class I-binding affinity. The types are modifications that can be made are listed below. Each modification represents a separate embodiment of the present invention.
  • In another embodiment, a peptide enumerated in the Examples of the present invention is further modified by mutating an anchor residue to an MHC class I preferred anchor residue, which can be, in other embodiments, any of the anchor residues enumerated herein. In another embodiment, a peptide of the present invention containing an MHC class I preferred anchor residue is further modified by mutating the anchor residue to a different MHC class I preferred residue for that location. The different preferred residue can be, in other embodiments, any of the preferred residues enumerated herein.
  • In another embodiment, the anchor residue that is further modified is in the 1 position. In another embodiment, the anchor residue is in the 2 position. In another embodiment, the anchor residue is in the 3 position. In another embodiment, the anchor residue is in the 4 position. In another embodiment, the anchor residue is in the 5 position. In another embodiment, the anchor residue is in the 6 position. In another embodiment, the anchor residue is in the 7 position. In another embodiment, the anchor residue is in the 8 position. In another embodiment, the anchor residue is in the 9 position. In the case of HLA class I-binding peptides, residues other than 2 and 9 can serve as secondary anchor residues; therefore, mutating them can improve MHC class I binding. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, a peptide of methods and compositions of the present invention is a length variant of a peptide enumerated in the Examples. In another embodiment, the length variant is one amino acid (AA) shorter than the peptide from the Examples. In another embodiment, the length variant is two AA shorter than the peptide from the Examples. In another embodiment, the length variant is more than two AA shorter than the peptide from the Examples. In another embodiment, the shorter peptide is truncated on the N-terminal end. In another embodiment, the shorter peptide is truncated on the C-terminal end. In another embodiment, the truncated peptide is truncated on both the N-terminal and C-terminal ends. Peptides are, in another embodiment, amenable to truncation without changing affinity for HLA molecules, as is well known in the art.
  • Each of the above truncated peptides represents a separate embodiment of the present invention.
  • In another embodiment, the length variant is longer than a peptide enumerated in the Examples of the present invention. In another embodiment, the longer peptide is extended on the N-terminal end in accordance with the surrounding WT1 sequence. Peptides are, in another embodiment, amenable to extension on the N-terminal end without changing affinity for HLA molecules, as is well known in the art. Such peptides are thus equivalents of the peptides enumerated in the Examples. In another embodiment, the N-terminal extended peptide is extended by one residue. In another embodiment, the N-terminal extended peptide is extended by two residues. In another embodiment, the N-terminal extended peptide is extended by three residues. In another embodiment, the N-terminal extended peptide is extended by more than three residues.
  • In another embodiment, the longer peptide is extended on the C terminal end in accordance with the surrounding WT1 sequence. Peptides are, in another embodiment, amenable to extension on the C-terminal end without changing affinity for HLA molecules, as is well known in the art. Such peptides are thus equivalents of the peptides enumerated in the Examples of the present invention. In another embodiment, the C-terminal extended peptide is extended by one residue. In another embodiment, the C-terminal extended peptide is extended by two residues. In another embodiment, the C-terminal extended peptide is extended by three residues. In another embodiment, the C-terminal extended peptide is extended by more than three residues.
  • In another embodiment, the extended peptide is extended on both the N-terminal and C-terminal ends in accordance with the surrounding WT1 sequence.
  • Each of the above extended peptides represents a separate embodiment of the present invention.
  • In another embodiment, a truncated peptide of the present invention retains the HLA anchor residues (e.g. the HLA class I anchor residues) on the second residue and the C-terminal residue, with a smaller number of intervening residues (e.g. 5) than a peptide enumerated in the Examples of the present invention. Peptides are, in another embodiment, amenable to such mutation without changing affinity for
  • HLA molecules. In another embodiment, such a truncated peptide is designed by removing one of the intervening residues of one of the above sequences. In another embodiment, the HLA anchor residues are retained on the second and eighth residues. In another embodiment, the HLA anchor residues are retained on the first and eighth residues. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, an extended peptide of the present invention retains the HLA anchor residues (e.g. the HLA class I anchor residues) on the second residue and the C-terminal residue, with a larger number of intervening residues (e.g. 7 or 8) than a peptide enumerated in the Examples of the present invention. In another embodiment, such an extended peptide is designed by adding one or more residues between two of the intervening residues of one of the above sequences. It is well known in the art that residues can be removed from or added between the intervening sequences of HLA-binding peptides without changing affinity for HLA. Such peptides are thus equivalents of the peptides enumerated in the Examples of the present invention. In another embodiment, the HLA anchor residues are retained on the second and ninth residues. In another embodiment, the HLA anchor residues are retained on the first and eighth residues. In another embodiment, the HLA anchor residues are retained on the two residues separated by six intervening residues. Each possibility represents a separate embodiment of the present invention.
  • “Fragment,” in another embodiment, refers to a peptide of 11 or more AA in length. In another embodiment, a peptide fragment of the present invention is 16 or more AA long. In another embodiment, the fragment is 12 or more AA long. In another embodiment, the fragment is 13 or more AA. In another embodiment, the fragment is 14 or more AA. In another embodiment, the fragment is 15 or more AA. In another embodiment, the fragment is 17 or more AA. In another embodiment, the fragment is 18 or more AA. In another embodiment, the fragment is 19 or more AA. In another embodiment, the fragment is 22 or more AA. In another embodiment, the fragment is 8-12 AA. In another embodiment, the fragment is about 8-12 AA. In another embodiment, the fragment is 16-19 AA. In another embodiment, the fragment is about 16-19 AA. In another embodiment, the fragment 10-25 AA. In another embodiment, the fragment is about 10-25 AA. In another embodiment, the fragment has any other length. Each possibility represents a separate embodiment of the present invention.
  • “Fragment of a WT1 protein,” in another embodiment, refers to any of the definitions of “fragment” found herein. Each definition represents a separate embodiment of the present invention.
  • In another embodiment, a peptide of the present invention is homologous to a peptide enumerated in the Examples. The terms “homology,” “homologous,” etc., when in reference to any protein or peptide, refer, in another embodiment, to a percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for the alignment are well known in the art.
  • In another embodiment, the term “homology,” when in reference to any nucleic acid sequence similarly indicates a percentage of nucleotides in a candidate sequence that are identical with the nucleotides of a corresponding native nucleic acid sequence.
  • Homology is, in another embodiment, determined by computer algorithm for sequence alignment, by methods well described in the art. In other embodiments, computer algorithm analysis of nucleic acid sequence homology includes the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages.
  • In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 70%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 75%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 78%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 82%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 87%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than [0128] 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 92%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 95%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 97%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 1-160, 162-185, 190, 191 or 193 of 100%. Each possibility represents a separate embodiment of the present invention. [00114] In another embodiment, homology is determined via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y). In another embodiments, methods of hybridization are carried out under moderate to stringent conditions, to the complement of a DNA encoding a native caspase peptide. Hybridization conditions being, for example, overnight incubation at 42° C. in a solution comprising: 10-20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.
  • Each of the above homologues and variants of peptides enumerated in the Examples represents a separate embodiment of the present invention.
  • In another embodiment, the present invention provides a composition comprising a peptide of this invention. In another embodiment, the composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the composition further comprises an adjuvant. In another embodiment, the composition comprises 2 or more peptides of the present invention. In another embodiment, the composition further comprises any of the additives, compounds, or excipients set forth hereinbelow. In another embodiment, the adjuvant is KLH, QS21, Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG or alum. In other embodiments, the carrier is any carrier enumerated herein. In other embodiments, the adjuvant is any adjuvant enumerated herein. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, this invention provides a vaccine comprising a peptide of this invention. In another embodiment, this invention provides a vaccine comprising an antigen-presenting cell (APC) and a peptide of this invention. In another embodiment, the vaccine further comprises a carrier. In another embodiment, the vaccine further comprises an adjuvant. In another embodiment, the vaccine further comprises an APC. In another embodiment, the vaccine further comprises a combination of more than 1 of an antigen, carrier, and/or APC. In another embodiment, the vaccine is a cell-based composition. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the term “vaccine” refers to a material or composition that, when introduced into a subject, provides a prophylactic or therapeutic response for a particular disease, condition, or symptom of same. In another embodiment, this invention comprises peptide-based vaccines, wherein the peptide comprises any embodiment listed herein, including immunomodulating compounds such as cytokines, adjuvants, etc.
  • In another embodiment, a vaccine of methods and compositions of the present invention further comprises an adjuvant. In another embodiment, the adjuvant is a Montanide. In another embodiment the adjuvant is Montanide ISA 51. Montanide ISA 51 contains a natural metabolizable oil and a refined emulsifier. In another embodiment, the adjuvant is GM-CSF. Recombinant GM-CSF is a human protein grown, in another embodiment, in a yeast (S. cerevisiae) vector. GM-CSF promotes clonal expansion and differentiation of hematopoietic progenitor cells, APC, and dendritic cells and T cells.
  • In another embodiment, the adjuvant is a cytokine. In another embodiment, the adjuvant is a growth factor. In another embodiment, the adjuvant is a cell population. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is Freund's incomplete adjuvant. In another embodiment, the adjuvant is aluminum phosphate. In another embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is BCG. In another embodiment, the adjuvant is alum.
  • In another embodiment, the adjuvant is an interleukin. In another embodiment, the adjuvant is a chemokine. In another embodiment, the adjuvant is any other type of adjuvant known in the art. In another embodiment, the WT1 vaccine comprises two the above adjuvants. In another embodiment, the WT1 vaccine comprises more than two the above adjuvants. Each possibility represents a separate embodiment of the present invention.
  • In other embodiments, a vaccine or composition of the present invention can comprise any of the embodiments of WT1 peptides of the present invention and combinations thereof. Each possibility represents a separate embodiment of the present invention.
  • It is to be understood that any embodiments described herein, regarding peptides, vaccines and compositions of this invention can be employed in any of the methods of this invention. Each combination of peptide, vaccine, or composition with a method represents an embodiment thereof.
  • In another embodiment, the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby treating a subject with a WT1-expressing cancer.
  • In another embodiment, the present invention provides a method of treating a subject with an MDS, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby treating a subject with an MDS.
  • In another embodiment, the present invention provides a method of suppressing or halting the progression of a WT1-expressing cancer in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby suppressing or halting the progression of a WT1-expressing cancer.
  • In another embodiment, the present invention provides a method of reducing the incidence of a WT1-expressing cancer in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of a WT1-expressing cancer in a subject.
  • In another embodiment, the present invention provides a method of reducing the incidence of an AML in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of an AML.
  • In another embodiment, the present invention provides a method of reducing the incidence of relapse of a WT1-expressing cancer in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of relapse of a WT1-expressing cancer in a subject.
  • In another embodiment, the present invention provides a method of reducing the incidence of relapse of an AML in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of relapse of an AML in a subject.
  • In another embodiment, the present invention provides a method of breaking a T cell tolerance of a subject to a WT1-expressing cancer, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby breaking a T cell tolerance to a WT1-expressing cancer.
  • In another embodiment, the present invention provides a method of treating a subject having a WT1-expressing cancer, comprising (a) inducing in a donor formation and proliferation of human cytotoxic T lymphocytes (CTL) that recognize a malignant cell of the cancer by a method of the present invention; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer.
  • In another embodiment, the present invention provides a method of treating a subject having a WT 1-expressing cancer, comprising (a) inducing ex vivo formation and proliferation of human CTL that recognize a malignant cell of the cancer by a method of the present invention, wherein the human immune cells are obtained from a donor; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer.
  • In another embodiment, the present invention provides a method of inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein; or a combination thereof. This method can be conducted in vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect.
  • Methods for ex vivo immunotherapy are well known in the art and are described, for example, in United States Patent Application Ser. Nos. 2006/0057130, 2005/0221481, 2005/0214268, 2003/0175272, 2002/0127718, and U.S. Pat. No. 5,229,115, which are incorporated herein by reference. Additional methods are well known in the art and are described, for example, in Davis I D et al (Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8+ T cells efficiently in cancer patients. J Immunother. 2006 September-October; 29(5):499-511) and Mitchell M S et al (The cytotoxic T cell response to peptide analogs of the HLA-A*0201-restricted MUC1 signal sequence epitope, M1.2. Cancer Immunol Immunother. 2006 Jul. 28). Each method represents a separate embodiment of the present invention.
  • In another embodiment, the present invention provides a method of inducing the formation and proliferation of CTL specific for cells of a WT1-expressing cancer, the method comprising contacting a lymphocyte population with a vaccine of the present invention. In another embodiment, the vaccine is an APC associated with a peptide of the present invention. In another embodiment, the vaccine is an APC associated with a mixture of peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, this invention provides a method of generating a heteroclitic immune response in a subject, wherein the heteroclitic immune response is directed against a WT1-expressing cancer, the method comprising administering to the subject a vaccine of the present invention, thereby generating a heteroclitic immune response.
  • In another embodiment, the present invention provides a method of inducing an anti-mesothelioma immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT1 protein; or (b) a fragment of a WT protein, thereby inducing an anti-mesothelioma immune response in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the present invention provides a method of inducing an anti-mesothelioma immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising a nucleotide molecule encoding (a) a WT1 protein; or (b) a fragment of a WT1 protein, thereby inducing an anti-mesothelioma immune response in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the present invention provides a method of treating a subject with a mesothelioma, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; or (b) a fragment of a WT protein, thereby treating a subject with a mesothelioma. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the present invention provides a method of treating a subject with a mesothelioma, the method comprising the step of administering to the subject an immunogenic composition comprising a nucleotide molecule encoding (a) a WT1 protein; or (b) a fragment of a WT1 protein, thereby treating a subject with a mesothelioma. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the present invention provides a method of reducing an incidence of a mesothelioma, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; or (b) a fragment of a WT protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the present invention provides a method of reducing an incidence of a mesothelioma, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising a nucleotide molecule encoding (a) a WT1 protein; or (b) a fragment of a WT1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, a target cell of an immune response elicited by a method of the present invention presents the WT1 peptide of the present invention, or a corresponding WT1 fragment, on an HLA molecule. In another embodiment, the HLA molecule is an HLA class I molecule. In other embodiments, the HLA molecule is any HLA class I subtype or HLA class I molecule known in the art. In another embodiment, the immune response against the WT1 peptide or fragment is a heteroclitic immune response. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the WT1-expressing cancer is an acute myelogenous leukemia (AML). In another embodiment, the WT1-expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT1-expressing cancer is an MDS. In another embodiment, the WT1-expressing cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the WT1-expressing cancer is a Wilms' tumor. In another embodiment, the WT1-expressing cancer is a leukemia. In another embodiment, the WT1-expressing cancer is a hematological cancer. In another embodiment, the WT1-expressing cancer is a lymphoma. In another embodiment, the WT1-expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT1-expressing cancer is a mesothelioma. In another embodiment, the WT1-expressing cancer is a malignant mesothelioma. In another embodiment, the WT1-expressing cancer is a gastric cancer. In another embodiment, the WT1-expressing cancer is a colon cancer. In another embodiment, the WT1-expressing cancer is a lung cancer. In another embodiment, the WT1-expressing cancer is abreast cancer. In another embodiment, the WT1-expressing cancer is a germ cell tumor. In another embodiment, the WT1-expressing cancer is an ovarian cancer. In another embodiment, the WT 1-expressing cancer is a uterine cancer. In another embodiment, the WT 1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a liver cancer. In another embodiment, the WT1-expressing cancer is a renal cancer. In another embodiment, the WT1-expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT1-expressing cancer is a sarcoma. In another embodiment, the WT1-expressing cancer is any other carcinoma or sarcoma.
  • In another embodiment, the WT1-expressing cancer is a solid tumor. In another embodiment, the solid tumor is associated with a WT1-expressing cancer. In another embodiment, the solid tumor is associated with a myelodysplastic syndrome (MDS). In another embodiment, the solid tumor is associated with a non-small cell lung cancer (NSCLC). In another embodiment, the solid tumor is associated with a lung cancer. In another embodiment, the solid tumor is associated with a breast cancer. In another embodiment, the solid tumor is associated with a colorectal cancer. In another embodiment, the solid tumor is associated with a prostate cancer. In another embodiment, the solid tumor is associated with an ovarian cancer. In another embodiment, the solid tumor is associated with a renal cancer. In another embodiment, the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma.
  • In another embodiment, a cancer or tumor treated by a method of the present invention is suspected to express WT1. In another embodiment, WT1 expression has not been verified by testing of the actual tumor sample. In another embodiment, the cancer or tumor is of a type known to express WT1 in many cases. In another embodiment, the type expresses WT1 in the majority of cases.
  • Each type of WT1-expressing cancer or tumor, and cancer or tumor suspected to express WT1, represents a separate embodiment of the present invention.
  • Any embodiments enumerated herein, regarding peptides, vaccines and compositions of this invention can be employed in any of the methods of this invention, and each represents an embodiment thereof.
  • In another embodiment, multiple peptides of this invention are used to stimulate an immune response in methods of the present invention.
  • The methods disclosed herein will be understood by those in the art to enable design of other WT1-derived peptides. The methods further enable design of peptides binding to other HLA molecules. The methods further enable design of vaccines combining WT1-derived peptides of the present invention. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, vaccines of the present invention have the advantage of activating or eliciting WT1-specific CD4+ T cells containing a variety of different HLA class II alleles. In another embodiment, the vaccines have the advantage of activating or eliciting WT1-specific CD4+ T cells in a substantial proportion of the population (e.g. in different embodiments, 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%, 90%, 95%, or greater than 95%). In another embodiment, the vaccines activate or elicit WT1-specific CD4+ T cells in a substantial proportion of a particular population (e.g. American Caucasians). Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, methods of the present invention provide for an improvement in an immune response that has already been mounted by a subject. In another embodiment, methods of the present invention comprise administering the peptide, composition, or vaccine 2 or more times. In another embodiment, the peptides are varied in their composition, concentration, or a combination thereof. In another embodiment, the peptides provide for the initiation of an immune response against an antigen of interest in a subject who has not yet initiated an immune response against the antigen. In another embodiment, the CTL that are induced proliferate in response to presentation of the peptide on the APC or cancer cell. In other embodiments, reference to modulation of the immune response involves, either or both the humoral and cell-mediated arms of the immune system, which is accompanied by the presence of Th2 and Th1 T helper cells, respectively, or in another embodiment, each arm individually.
  • In other embodiments, the methods affecting the growth of a tumor result in (1) the direct inhibition of tumor cell division, or (2) immune cell mediated tumor cell lysis, or both, which leads to a suppression in the net expansion of tumor cells.
  • Inhibition of tumor growth by either of these two mechanisms can be readily determined by one of ordinary skill in the art based upon a number of well-known methods. In another embodiment, tumor inhibition is determined by measuring the actual tumor size over a period of time. In another embodiment, tumor inhibition can be determined by estimating the size of a tumor (over a period of time) utilizing methods well known to those of skill in the art. More specifically, a variety of radiologic imaging methods (e.g., single photon and positron emission computerized tomography; see generally, “Nuclear Medicine in Clinical Oncology,” Winkler, C. (ed.) Springer-Verlag, New York, 1986), can be utilized to estimate tumor size. Such methods can also utilize a variety of imaging agents, including for example, conventional imaging agents (e.g., Gallium-67 citrate), as well as specialized reagents for metabolite imaging, receptor imaging, or immunologic imaging (e.g., radiolabeled monoclonal antibody specific tumor markers). In addition, non-radioactive methods such as ultrasound (see, “Ultrasonic Differential Diagnosis of Tumors”, Kossoff and Fukuda, (eds.), Igaku-Shoin, New York, 1984), can also be utilized to estimate the size of a tumor.
  • In addition to the in vivo methods for determining tumor inhibition discussed above, a variety of in vitro methods can be utilized in order to predict in vivo tumor inhibition. Representative examples include lymphocyte mediated anti-tumor cytolytic activity determined for example, by a 51Cr release assay (Examples), tumor dependent lymphocyte proliferation (Ioannides, et al., J. Immunol. 146(5):1700-1707, 1991), in vitro generation of tumor specific antibodies (Herlyn, et al., J. Immunol. Meth. 73:157-167, 1984), cell (e.g., CTL, helper T-cell) or humoral (e.g., antibody) mediated inhibition of cell growth in vitro (Gazit, et al., Cancer Immunol Immunother 35:135-144, 1992), and, for any of these assays, determination of cell precursor frequency (Vose, Int. J. Cancer 30:135-142 (1982), and others.
  • In another embodiment, methods of suppressing tumor growth indicate a growth state that is curtailed compared to growth without contact with, or exposure to a peptide of this invention. Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a 3H-thymidine incorporation assay, or counting tumor cells. “Suppressing” tumor cell growth refers, in other embodiments, to slowing, delaying, or stopping tumor growth, or to tumor shrinkage. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment of methods and compositions of the present invention, WT1 expression is measured. In another embodiment, WT1 transcript expression is measured. In another embodiment, WT1 protein levels in the tumor are measured. Each possibility represents a separate embodiment of the present invention.
  • Methods of determining the presence and magnitude of an immune response are well known in the art. In another embodiment, lymphocyte proliferation assays, wherein T cell uptake of a radioactive substance, e.g. 3H-thymidine is measured as a function of cell proliferation. In other embodiments, detection of T cell proliferation is accomplished by measuring increases in interleukin-2 (IL-2) production, Ca2+ flux, or dye uptake, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, CTL stimulation is determined by means known to those skilled in the art, including, detection of cell proliferation, cytokine production and others. Analysis of the types and quantities of cytokines secreted by T cells upon contacting ligand-pulsed targets can be a measure of functional activity. Cytokines can be measured by ELISA or ELISPOT assays to determine the rate and total amount of cytokine production. (Fujihashi K. et al. (1993) J. Immunol. Meth. 160: 181; Tanguay S. and Killion J. J. (1994) Lymphokine Cytokine Res. 13:259).
  • In another embodiment, CTL activity is determined by 51Cr-release lysis assay. Lysis of peptide-pulsed 51Cr-labeled targets by antigen-specific T cells can be compared for target cells pulsed with control peptide. In another embodiment, T cells are stimulated with a peptide of this invention, and lysis of target cells expressing the native peptide in the context of MHC can be determined. The kinetics of lysis as well as overall target lysis at a fixed timepoint (e.g., 4 hours) are used, in another embodiment, to evaluate ligand performance (Ware C. F. et al. (1983) J Immunol 131: 1312).
  • Methods of determining affinity of a peptide for an HLA molecule are well known in the art. In another embodiment, affinity is determined by TAP stabilization assays.
  • In another embodiment, affinity is determined by competition radioimmunoassay. In another embodiment, the following protocol is utilized: Target cells are washed two times in PBS with 1% bovine serum albumin (BSA; Fisher Chemicals, Fairlawn, N.J.). Cells are resuspended at 107/ml on ice, and the native cell surface bound peptides are stripped for 2 minutes at 0[deg.] C using citrate-phosphate buffer in the presence of 3 mg/ml beta2 microglobulin. The pellet is resuspended at 5×106 cells/ml in PBS/1% BSA in the presence of 3 mg/ml beta2 microglobulin and 30 mg/ml deoxyribonuclease, and 200 ml aliquots are incubated in the presence or absence of HLA-specific peptides for 10 min at 20° C., then with 125I-labeled peptide for 30 min at 20° C. Total bound 125I is determined after two washes with PBS/2% BSA and one wash with PBS. Relative affinities are determined by comparison of escalating concentrations of the test peptide versus a known binding peptide.
  • In another embodiment, a specificity analysis of the binding of peptide to HLA on surface of live cells (e.g. SKLY-16 cells) is conducted to confirm that the binding is to the appropriate HLA molecule and to characterize its restriction. This includes, in another embodiment, competition with excess unlabeled peptides known to bind to the same or disparate HLA molecules and use of target cells which express the same or disparate HLA types. This assay is performed, in another embodiment, on live fresh or 0.25% paraformaldehyde-fixed human PBMC, leukemia cell lines and EBV-transformed T-cell lines of specific HLA types. The relative avidity of the peptides found to bind MHC molecules on the specific cells are assayed by competition assays as described above against 125I-labeled peptides of known high affinity for the relevant HLA molecule, e.g., tyrosinase or HBV peptide sequence. In another embodiment, an HLA class II-binding peptide of methods and compositions of the present invention is longer than the minimum length for binding to an HLA class II molecule, which is, in another embodiment, about 12 AA. In another embodiment, increasing the length of the HLA class II-binding peptide enables binding to more than one HLA class II molecule. In another embodiment, increasing the length enables binding to an HLA class II molecule whose binding motif is not known. In another embodiment, increasing the length enables binding to an HLA class I molecule. In another embodiment, the binding motif of the HLA class I molecule is known. In another embodiment, the binding motif of the HLA class I molecule is not known. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the peptides utilized in methods and compositions of the present invention comprise a non-classical amino acid such as: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991) J. Am Chem. Soc. 113:2275-2283); (2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine (Kazmierski and Hruby (1991) Tetrahedron Lett. 32(41): 5769-5772); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis (1989) Ph.D. Thesis, University of Arizona); hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al. (1984) J. Takeda Res. Labs. 43:53-76) histidine isoquinoline carboxylic acid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131-138); and HIC (histidine cyclic urea), (Dharanipragada et al. (1993) Int. J. Pep. Protein Res. 42(1):68-77) and ((1992) Acta. Crst., Crystal Struc. Comm 48(IV): 1239-124).
  • In another embodiment, a peptide of this invention comprises an AA analog or peptidomimetic, which, in other embodiments, induces or favors specific secondary structures. Such peptides comprise, in other embodiments, the following: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a [beta]-turn inducing dipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838); [beta]-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5081-5082); [beta]-turn inducing analogs (Kemp et al. (1988) Tetrahedron Left. 29:5057-5060); alpha-helix inducing analogs (Kemp et al. (1988) Tetrahedron Left. 29:4935-4938); gamma-turn inducing analogs (Kemp et al. (1989) J. Org. Chem. 54:109:115); analogs provided by the following references: Nagai and Sato (1985) Tetrahedron Left. 26:647-650; and DiMaio et al. (1989) J. Chem. Soc. Perkin Trans, p. 1687; a Gly-Ala turn analog (Kahn et al. (1989) Tetrahedron Lett. 30:2317); amide bond isostere (Jones et al. (1988) Tetrahedron Left. 29(31):3853-3856); tretrazol (Zabrocki et al. (1988) J. Am. Chem. Soc. 110:5875-5880); DTC (Samanen et al. (1990) Int. J. Protein Pep. Res. 35:501:509); and analogs taught in Olson et al. (1990) J. Am. Chem. Sci. 112:323-333 and Garvey et al. (1990) J. Org. Chem. 55(3):936-940. Conformationally restricted mimetics of beta turns and beta bulges, and peptides containing them, are described in U.S. Pat. No. 5,440,013, issued Aug. 8, 1995 to Kahn.
  • In other embodiments, a peptide of this invention is conjugated to one of various other molecules, as described hereinbelow, which can be via covalent or non-covalent linkage (complexed), the nature of which varies, in another embodiment, depending on the particular purpose. In another embodiment, the peptide is covalently or non-covalently complexed to a macromolecular carrier, (e.g. an immunogenic carrier), including, but not limited to, natural and synthetic polymers, proteins, polysaccharides, polypeptides (amino acids), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids. In another embodiment, a peptide of this invention is linked to a substrate. In another embodiment, the peptide is conjugated to a fatty acid, for introduction into a liposome (U.S. Pat. No. 5,837,249). In another embodiment, a peptide of the invention is complexed covalently or non-covalently with a solid support, a variety of which are known in the art. In another embodiment, linkage of the peptide to the carrier, substrate, fatty acid, or solid support serves to increase an elicited an immune response.
  • In other embodiments, the carrier is thyroglobulin, an albumin (e.g. human serum albumin), tetanus toxoid, polyamino acids such as poly (lysine: glutamic acid), an influenza protein, hepatitis B virus core protein, keyhole limpet hemocyanin, an albumin, or another carrier protein or carrier peptide; hepatitis B virus recombinant vaccine, or an APC. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the term “amino acid” (AA) refers to a natural or, in another embodiment, an unnatural or synthetic AA, and can include, in other embodiments, glycine, D- or L optical isomers, AA analogs, peptidomimetics, or combinations thereof.
  • In another embodiment, the terms “cancer,” “neoplasm,” “neoplastic” or “tumor,” are used interchangeably and refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but also any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. In another embodiment, a tumor is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation, and in another embodiment, is identified by biochemical or immunologic findings, the latter which is used to identify cancerous cells, as well, in other embodiments.
  • Methods for synthesizing peptides are well known in the art. In another embodiment, the peptides of this invention are synthesized using an appropriate solid-state synthetic procedure (see for example, Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco, Calif. (1968); Merrifield (1967) Recent Progress in Hormone Res 23: 451). The activity of these peptides is tested, in other embodiments, using assays as described herein.
  • In another embodiment, the peptides of this invention are purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. In another embodiment, immuno-affinity chromatography is used, whereby an epitope is isolated by binding it to an affinity column comprising antibodies that were raised against that peptide, or a related peptide of the invention, and were affixed to a stationary support.
  • In another embodiment, affinity tags such as hexa-His (Invitrogen), Maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991) Meth. Enzymol. 194:508-509), glutathione-S-transferase, or others, are attached to the peptides of this invention to allow easy purification by passage over an appropriate affinity column. Isolated peptides can also be physically characterized, in other embodiments, using such techniques as proteolysis, nuclear magnetic resonance, and x-ray crystallography.
  • In another embodiment, the peptides of this invention are produced by in vitro translation, through known techniques, as will be evident to one skilled in the art. In another embodiment, the peptides are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand, (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320).
  • In another embodiment, the peptides of this invention further comprise a detectable label, which in another embodiment, is fluorescent, or in another embodiment, luminescent, or in another embodiment, radioactive, or in another embodiment, electron dense. In other embodiments, the detectable label comprises, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-galactosidase, luciferase, 32P, 125I, 3H and 14C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone, luciferin or any number of other such labels known to one skilled in the art. The particular label used will depend upon the type of immunoassay used.
  • In another embodiment, a peptide of this invention is linked to a substrate, which, in another embodiment, serves as a carrier. In another embodiment, linkage of the peptide to a substrate serves to increase an elicited an immune response.
  • In another embodiment, peptides of this invention are linked to other molecules, as described herein, using conventional cross-linking agents such as carbodiimide. Examples of carbodiimide are 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and 1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.
  • In other embodiments, the cross-linking agents comprise cyanogen bromide, glutaraldehyde and succinic anhydride. In general, any of a number of homo-bifunctional agents including a homo-bifunctional aldehyde, a homo-bifunctional epoxide, a homo-bifunctional imido-ester, a homo-bifunctional N-hydroxysuccinimide ester, a homo-bifunctional maleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyl disulfide, a homo-bifunctional aryl halide, a homo-bifunctional hydrazide, a homo-bifunctional diazonium derivative and a homo-bifunctional photoreactive compound can be used. Also envisioned, in other embodiments, are hetero-bifunctional compounds, for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine-reactive and a photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl-reactive group.
  • In other embodiments, the homo-bifunctional cross-linking agents include the bifunctional N-hydroxysuccinimide esters dithiobis(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imido-esters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers 1,4-di-[3′-(2′-pyridyldithio)propionamido]butane, bismaleimidohexane, and bis-N-maleimido-1,8-octane; the bifunctional aryl halides 1,5-difluoro-2,4-dinitrobenzene and 4,4′-difluoro-3,3′-dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[b-(4-azidosalicylamido)ethyl]disulfide; the bifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as 1,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipic acid dihydrazide, carbohydrazide, and succinic acid dihydrazide; the bifunctional diazoniums o-tolidine, diazotized and bis-diazotized benzidine; the bifunctional alkylhalides N,N′-ethylene-bis(iodoacetamide), N1N′-hexamethylene-bis(iodoacetamide), N,N′-undecamethylene-bis(iodoacetamide), as well as benzylhalides and halomustards, such as a 1a′-diiodo-p-xylene sulfonic acid and tri(2-chloroethyl)amine, respectively,
  • In other embodiments, hetero-bifunctional cross-linking agents used to link the peptides to other molecules, as described herein, include, but are not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MB S (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS (N-(.gamma-maleimidobutyryloxy)succmimide ester), MPBH (4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N-maleimidomethyl) cyclohexane-1-carboxyl-hydrazide), SMPT (succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene), and SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate).
  • In another embodiment, the peptides of the invention are formulated as non-covalent attachment of monomers through ionic, adsorptive, or biospecific interactions. Complexes of peptides with highly positively or negatively charged molecules can be accomplished, in another embodiment, through salt bridge formation under low ionic strength environments, such as in deionized water. Large complexes can be created, in another embodiment, using charged polymers such as poly-(L-glutamic acid) or poly-(L-lysine), which contain numerous negative and positive charges, respectively. In another embodiment, peptides are adsorbed to surfaces such as microparticle latex beads or to other hydrophobic polymers, of D-biotin (NHS-biotin), which reacts with available amine groups.
  • In another embodiment, a peptide of the present invention is linked to a carrier. In another embodiment, the carrier is KLH. In other embodiments, the carrier is any other carrier known in the art, including, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly (lysine:glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the peptides of this invention are conjugated to a lipid, such as P3 CSS. In another embodiment, the peptides of this invention are conjugated to a bead.
  • In another embodiment, the compositions of this invention further comprise immunomodulating compounds. In other embodiments, the immunomodulating compound is a cytokine, chemokine, or complement component that enhances expression of immune system accessory or adhesion molecules, their receptors, or combinations thereof. In some embodiments, the immunomodulating compound include interleukins, for example interleukins 1 to 15, interferons alpha, beta or gamma, tumour necrosis factor, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, macrophage inflammatory peptides MIP-Ia and MIP-Ib, complement components, or combinations thereof. In other embodiments, the immunomodulating compound stimulate expression, or enhanced expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-1, 2 or 3, cytokine receptors, or combination thereof.
  • In another embodiment, the immunomodulatory compound induces or enhances expression of co-stimulatory molecules that participate in the immune response, which include, in some embodiments, CD40 or its ligand, CD28, CTLA-4 or a B7 molecule. In another embodiment, the immunomodulatory compound induces or enhances expression of a heat stable antigen (HSA) (Liu Y. et al. (1992) J. Exp. Med. 175:437-445), chondroitin sulfate-modified MHC invariant chain (Ii-CS) (Naujokas M. F. et al (1993) Cell 74:257-268), or an intracellular adhesion molecule 1 (ICAM-I) (Van R. H. (1992) Cell 71: 1065-1068), which assists, in another embodiment, co-stimulation by interacting with their cognate ligands on the T cells.
  • In another embodiment, the composition comprises a solvent, including water, dispersion media, cell culture media, isotonic agents and the like. In another embodiment, the solvent is an aqueous isotonic buffered solution with a pH of around 7.0. In another embodiment, the composition comprises a diluent such as water, phosphate buffered saline, or saline. In another embodiment, the composition comprises a solvent, which is non-aqueous, such as propyl ethylene glycol, polyethylene glycol and vegetable oils.
  • In another embodiment, the composition is formulated for administration by any of the many techniques known to those of skill in the art. For example, this invention provides for administration of the pharmaceutical composition parenterally, intravenously, subcutaneously, intradermally, intramucosally, topically, orally, or by inhalation.
  • In another embodiment, the vaccine comprising a peptide of this invention further comprises a cell population, which, in another embodiment, comprises lymphocytes, monocytes, macrophages, dendritic cells, endothelial cells, stem cells or combinations thereof, which, in another embodiment are autologous, syngeneic or allogeneic, with respect to each other. In another embodiment, the cell population comprises a peptide of the present invention. In another embodiment, the cell population takes up the peptide. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the cell populations of this invention are obtained from in vivo sources, such as, for example, peripheral blood, leukopheresis blood product, apheresis blood product, peripheral lymph nodes, gut associated lymphoid tissue, spleen, thymus, cord blood, mesenteric lymph nodes, liver, sites of immunologic lesions, e.g. synovial fluid, pancreas, cerebrospinal fluid, tumor samples, granulomatous tissue, or any other source where such cells can be obtained. In another embodiment, the cell populations are obtained from human sources, which are, in other embodiments, from human fetal, neonatal, child, or adult sources. In another embodiment, the cell populations of this invention are obtained from animal sources, such as, for example, porcine or simian, or any other animal of interest. In another embodiment, the cell populations of this invention are obtained from subjects that are normal, or in another embodiment, diseased, or in another embodiment, susceptible to a disease of interest.
  • In another embodiment, the cell populations of this invention are separated via affinity-based separation methods. Techniques for affinity separation include, in other embodiments, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or use in conjunction with a monoclonal antibody, for example, complement and cytotoxins, and “panning” with an antibody attached to a solid matrix, such as a plate, or any other convenient technique. In other embodiment, separation techniques include the use of fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. In other embodiments, any technique that enables separation of the cell populations of this invention can be employed, and is to be considered as part of this invention.
  • In another embodiment, the dendritic cells are from the diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues, qualified as such (Steinman (1991) Ann. Rev. Immunol. 9:271-296). In another embodiment, the dendritic cells used in this invention are isolated from bone marrow, or in another embodiment, derived from bone marrow progenitor cells, or, in another embodiment, from isolated from/derived from peripheral blood, or in another embodiment, derived from, or are a cell line.
  • In another embodiment, the cell populations described herein are isolated from the white blood cell fraction of a mammal, such as a murine, simian or a human (See, e.g., WO 96/23060). The white blood cell fraction can be, in another embodiment, isolated from the peripheral blood of the mammal.
  • Methods of isolating dendritic cells are well known in the art. In another embodiment, the DC are isolated via a method which includes the following steps: (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the art such as leukophoresis; (b) separating the white blood cell fraction of step (a) into four or more subfractions by countercurrent centrifugal elutriation; (c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSF and IL-4, (d) identifying the dendritic cell-enriched fraction from step (c); and (e) collecting the enriched fraction of step (d), preferably at about 4[deg.] C.
  • In another embodiment, the dendritic cell-enriched fraction is identified by fluorescence-activated cell sorting, which identifies at least one of the following markers: HLA-DR, HLA-DQ, or B7.2, and the simultaneous absence of the following markers: CD3, CD14, CD16, 56, 57, and CD 19, 20.
  • In another embodiment, the cell population comprises lymphocytes, which are, in another embodiment, T cells, or in another embodiment, B cells. The T cells are, in other embodiments, characterized as NK cells, helper T cells, cytotoxic T lymphocytes (CTL), TBLs, native T cells, or combinations thereof. It is to be understood that T cells which are primary, or cell lines, clones, etc. are to be considered as part of this invention. In another embodiment, the T cells are CTL, or CTL lines, CTL clones, or CTLs isolated from tumor, inflammatory, or other infiltrates.
  • In another embodiment, hematopoietic stem or early progenitor cells comprise the cell populations used in this invention. In another embodiment, such populations are isolated or derived, by leukaphoresis. In another embodiment, the leukapheresis follows cytokine administration, from bone marrow, peripheral blood (PB) or neonatal umbilical cord blood. In another embodiment, the stem or progenitor cells are characterized by their surface expression of the surface antigen marker known as CD34+, and exclusion of expression of the surface lineage antigen markers, Lin−.
  • In another embodiment, the subject is administered a peptide, composition or vaccine of this invention, in conjunction with bone marrow cells. In another embodiment, the administration together with bone marrow cells embodiment follows previous irradiation of the subject, as part of the course of therapy, in order to suppress, inhibit or treat cancer in the subject.
  • In another embodiment, the phrase “contacting a cell” or “contacting a population” refers to a method of exposure, which can be, in other embodiments, direct or indirect. In another embodiment, such contact comprises direct injection of the cell through any means well known in the art, such as microinjection. It is also envisaged, in another embodiment, that supply to the cell is indirect, such as via provision in a culture medium that surrounds the cell, or administration to a subject, via any route well known in the art, and as described herein.
  • In another embodiment, CTL generation of methods of the present invention is accomplished in vivo, and is effected by introducing into a subject an antigen presenting cell contacted in vitro with a peptide of this invention (See for example Paglia et al. (1996) J. Exp. Med. 183:317-322).
  • In another embodiment, the peptides of methods and compositions of the present invention are delivered to APC. In another embodiment, the peptide-pulsed APC are administered to a subject to elicit and immune response or treat or inhibit growth or recurrence of a tumor. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the peptides are delivered to APC in the form of cDNA encoding the peptides. In another embodiment, the term “antigen-presenting cells” (APC) refers to dendritic cells (DC), monocytes/macrophages, B lymphocytes or other cell type(s) expressing the necessary MHC/co-stimulatory molecules, which effectively allow for T cell recognition of the presented peptide. In another embodiment, the APC is a cancer cell. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the CTL are contacted with 2 or more APC populations. In another embodiment, the 2 or more APC populations present different peptides. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, techniques that lead to the expression of antigen in the cytosol of APC (e.g. DC) are used to deliver the peptides to the APC. Methods for expressing antigens on APC are well known in the art. In another embodiment, the techniques include (1) the introduction into the APC of naked DNA encoding a peptide of this invention, (2) infection of APC with recombinant vectors expressing a peptide of this invention, and (3) introduction of a peptide of this invention into the cytosol of an APC using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med. 184:465-472; Rouse et al. (1994) J. Virol. 68:5685-5689; and Nair et al. (1992) J. Exp. Med. 175:609-612).
  • In another embodiment, foster APC such as those derived from the human cell line 174×CEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763-1771), are used, as exemplified herein.
  • In another embodiment, as described herein, the subject is exposed to a peptide, or a composition/cell population comprising a peptide of this invention, which differs from the native protein expressed, wherein subsequently a host immune cross-reactive with the native protein/antigen develops.
  • In another embodiment, the subject, as referred to in any of the methods or embodiments of this invention is a human. In other embodiments, the subject is a mammal, which can be a mouse, rat, rabbit, hamster, guinea pig, horse, cow, sheep, goat, pig, cat, dog, monkey, or ape. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, peptides, vaccines, and compositions of this invention stimulate an immune response that results in tumor cell lysis.
  • In another embodiment, any of the methods described herein is used to elicit CTL, which are elicited in vitro. In another embodiment, the CTL are elicited ex-vivo. In another embodiment, the CTL are elicited in vitro. The resulting CTL, are, in another embodiment, administered to the subject, thereby treating the condition associated with the peptide, an expression product comprising the peptide, or a homologue thereof. Each possibility represents a separate embodiment of the present invention.
  • In another embodiment, the method entails introduction of the genetic sequence that encodes the peptides of this invention using, e.g., one or more nucleic acid delivery techniques. Nucleic acids of the invention include, in another embodiment, DNA, RNA and mixtures of DNA and RNA, alone or in conjunction with non-nucleic acid components. In another embodiment, the method comprises administering to the subject a vector comprising a nucleotide sequence, which encodes a peptide of the present invention (Tindle, R. W. et al. Virology (1994) 200:54). In another embodiment, the method comprises administering to the subject naked DNA which encodes a peptide, or in another embodiment, two or more peptides of this invention (Nabel, et al. PNAS-USA (1990) 90: 11307). In another embodiment, multi-epitope, analogue-based cancer vaccines are utilized (Fikes et al, Design of multi-epitope, analogue-based cancer vaccines. Expert Opin Biol Ther. 2003 September; 3(6):985-93). Each possibility represents a separate embodiment of the present invention.
  • Nucleic acids can be administered to a subject via any means as is known in the art, including parenteral or intravenous administration, or in another embodiment, by means of a gene gun. In another embodiment, the nucleic acids are administered in a composition, which correspond, in other embodiments, to any embodiment listed herein.
  • Vectors for use according to methods of this invention can comprise any vector that facilitates or allows for the expression of a peptide of this invention. Vectors comprise, in some embodiments, attenuated viruses, such as vaccinia or fowlpox, such as described in, e.g., U.S. Pat. No. 4,722,848, incorporated herein by reference. In another embodiment, the vector is BCG (Bacille Calmette Guerin), such as described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g., Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein.
  • In another embodiment, the vector further encodes for an immunomodulatory compound, as described herein. In another embodiment, the subject is administered an additional vector encoding same, concurrent, prior to or following administration of the vector encoding a peptide of this invention to the subject.
  • In another embodiment, the peptides, compositions and vaccines of this invention are administered to a subject, or utilized in the methods of this invention, in combination with other anticancer compounds and chemotherapeutics, including monoclonal antibodies directed against alternate cancer antigens, or, in another embodiment, epitopes that consist of an AA sequence which corresponds to, or in part to, that from which the peptides of this invention are derived.
  • Various embodiments of dosage ranges are contemplated by this invention. In another embodiment, the dosage is 20 μg per peptide per day. In another embodiment, the dosage is 10 μg/peptide/day. In another embodiment, the dosage is 30 μg/peptide/day. In another embodiment, the dosage is 40 μg/peptide/day. In another embodiment, the dosage is 60 μg/peptide/day. In another embodiment, the dosage is 80 μg/peptide/day. In another embodiment, the dosage is 100 μg/peptide/day. In another embodiment, the dosage is 150 μg/peptide/day. In another embodiment, the dosage is 200 μg/peptide/day. In another embodiment, the dosage is 300 μg/peptide/day. In another embodiment, the dosage is 400 μg/peptide/day. In another embodiment, the dosage is 600 μg/peptide/day. In another embodiment, the dosage is 800 μg/peptide/day. In another embodiment, the dosage is 1000 μg/peptide/day. In another embodiment, the dosage is 1500 μg/peptide/day. In another embodiment, the dosage is 2000 μg/peptide/day.
  • In another embodiment, the dosage is 10 μg/peptide/dose. In another embodiment, the dosage is 30 μg/peptide/dose. In another embodiment, the dosage is 40 μg/peptide/dose. In another embodiment, the dosage is 60 μg/peptide/dose. In another embodiment, the dosage is 80 μg/peptide/dose. In another embodiment, the dosage is 100 μg/peptide/dose. In another embodiment, the dosage is 150 μg/peptide/dose. In another embodiment, the dosage is 200 μg/peptide/dose. In another embodiment, the dosage is 300 μg/peptide/dose. In another embodiment, the dosage is 400 μg/peptide/dose. In another embodiment, the dosage is 600 μg/peptide/dose. In another embodiment, the dosage is 800 μg/peptide/dose. In another embodiment, the dosage is 1000 μg/peptide/dose. In another embodiment, the dosage is 1500 μg/peptide/dose. In another embodiment, the dosage is 2000 μg/peptide/dose.
  • In another embodiment, the dosage is 10-20 μg/peptide/dose. In another embodiment, the dosage is 20-30 μg/peptide/dose. In another embodiment, the dosage is 20-40 μg/peptide/dose. In another embodiment, the dosage is 30-60 μg/peptide/dose. In another embodiment, the dosage is 40-80 μg/peptide/dose. In another embodiment, the dosage is 50-100 μg/peptide/dose. In another embodiment, the dosage is 50-150 μg/peptide/dose. In another embodiment, the dosage is 100-200 μg/peptide/dose. In another embodiment, the dosage is 200-300 μg/peptide/dose. In another embodiment, the dosage is 300-400 μg/peptide/dose. In another embodiment, the dosage is 400-600 μg/peptide/dose. In another embodiment, the dosage is 500-800 μg/peptide/dose. In another embodiment, the dosage is 800-1000 μg/peptide/dose. In another embodiment, the dosage is 1000-1500 μg/peptide/dose. In another embodiment, the dosage is 1500-2000 μg/peptide/dose.
  • In another embodiment, the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts.
  • Each of the above doses represents a separate embodiment of the present invention.
  • Various embodiments of dosage ranges are contemplated by this invention. In another embodiment, the dosage is 20 mg per peptide per day. In another embodiment, the dosage is 10 mg/peptide/day. In another embodiment, the dosage is 30 mg/peptide/day. In another embodiment, the dosage is 40 mg/peptide/day. In another embodiment, the dosage is 60 mg/peptide/day. In another embodiment, the dosage is 80 mg/peptide/day. In another embodiment, the dosage is 100 mg/peptide/day. In another embodiment, the dosage is 150 mg/peptide/day. In another embodiment, the dosage is 200 mg/peptide/day. In another embodiment, the dosage is 300 mg/peptide/day. In another embodiment, the dosage is 400 mg/peptide/day. In another embodiment, the dosage is 600 mg/peptide/day. In another embodiment, the dosage is 800 mg/peptide/day. In another embodiment, the dosage is 1000 mg/peptide/day.
  • In another embodiment, the dosage is 10 mg/peptide/dose. In another embodiment, the dosage is 30 mg/peptide/dose. In another embodiment, the dosage is 40 mg/peptide/dose. In another embodiment, the dosage is 60 mg/peptide/dose. In another embodiment, the dosage is 80 mg/peptide/dose. In another embodiment, the dosage is 100 mg/peptide/dose. In another embodiment, the dosage is 150 mg/peptide/dose. In another embodiment, the dosage is 200 mg/peptide/dose. In another embodiment, the dosage is 300 mg/peptide/dose. In another embodiment, the dosage is 400 mg/peptide/dose. In another embodiment, the dosage is 600 mg/peptide/dose. In another embodiment, the dosage is 800 mg/peptide/dose. In another embodiment, the dosage is 1000 mg/peptide/dose.
  • In another embodiment, the dosage is 10-20 mg/peptide/dose. In another embodiment, the dosage is 20-30 mg/peptide/dose. In another embodiment, the dosage is 20-40 mg/peptide/dose. In another embodiment, the dosage is 30-60 mg/peptide/dose. In another embodiment, the dosage is 40-80 mg/peptide/dose. In another embodiment, the dosage is 50-100 mg/peptide/dose. In another embodiment, the dosage is 50-150 mg/peptide/dose. In another embodiment, the dosage is 100-200 mg/peptide/dose. In another embodiment, the dosage is 200-300 mg/peptide/dose. In another embodiment, the dosage is 300-400 mg/peptide/dose. In another embodiment, the dosage is 400-600 mg/peptide/dose. In another embodiment, the dosage is 500-800 mg/peptide/dose. In another embodiment, the dosage is 800-1000 mg/peptide/dose.
  • In another embodiment, the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts.
  • Each of the above doses represents a separate embodiment of the present invention.
  • In another embodiment, the present invention provides a kit comprising a peptide, composition or vaccine of the present invention. In another embodiment, the kit further comprises a label or packaging insert. In another embodiment, the kit is used for detecting a WT1-specific CD4 response through the use of a delayed-type hypersensitivity test. In another embodiment, the kit is used for any other method enumerated herein. In another embodiment, the kit is used for any other method known in the art. Each possibility represents a separate embodiment of the present invention.
  • Among those antigens uniquely or differentially expressed by malignant cells, WT1 is considered one of the most promising (47). However, the number of immunogenic WT1 peptide antigens previously identified and reported is very limited, and largely confined to a set of peptides presented by the HLA alleles A0201, A2402 and DRB10401. As will be seen from the examples presented below, using a pool of overlapping 15-mer peptides spanning the amino acid sequence of WT1 loaded on autologous APCs for sensitization, WT1 peptide-specific IFNγ+CD4+ and CD8 T-cell responses were generated from the blood of 41/56 (78%) normal donors, and thereafter the epitopes eliciting these responses and their presenting HLA alleles were identified. Of the 42 WT1 peptide antigens described, all but one have not been heretofore identified. The new immunogenic peptides identified include 36 peptides presented by class I HLA alleles and 5 presented by class II HLA alleles. Of the peptides presented by class I HLA alleles, 10 nonamer epitopes were identified which could be presented by from 2-4 different HLA alleles. Also identified, within 4 pentadecapeptides, were overlapping 11-mer and nonamer sequences that co-induced distinguishable CD4+ IFNγ+ and CD8+ IFNγ+ T-cells. Whether and to what degree epitopes that can be presented by more than one allele can elicit enhanced WT1 specific responses in individuals inheriting both presenting HLA alleles or both the class I and class II presenting HLA alleles in those instances in which overlapping sequences are contained in the same 15-mer is readily determinable; however, inclusion of such peptides in WT1 vaccines could significantly broaden their applicability particularly among patients not inheriting HLA-A0201 or A2402.
  • As shown in the examples, those peptides presented by class I HLA alleles elicited IFNγ+CD8+ T-cells that were able to lyse peptide loaded autologous APCs as well as allogeneic APCs sharing the T-cells' restricting HLA allele in 50/51 (99%) and 48/51 (94%)cultures tested respectively (Table 1, 2). More importantly, of 36 HLA-restricted WT1 peptide specific T-cell lines that could be tested, T-cell lines specific for 29 epitopes including 2/4 epitopes presented by class II and 27/32 presented by class I alleles, were also able to lyse WT1+ leukemic blasts sharing the T-cells' restricting HLA allele. The failure of the HLA-restricted WT1 epitope-specific T-cells to lyse allogeneic PHA blasts from the same leukemic patients (Table 3A), coupled with the differential leukemocidal activity of T-cells sensitized with WT1 peptide-loaded autologous EBVBLCL when compared to aliquots of the same T-cells sensitized with autologous EBVBLCL alone (Table 3B) indicates that the leukemocidal activity is WT1 peptide-specific and not a result of contaminating alloreactive T-cells. Thus, these data show that 29/36 immunogenic peptides of WT1 identified (80%) can be processed and presented by WT1+ leukemic cells at concentrations adequate for WT1 epitope-specific T-cell recognition and cytolysis.
  • In FIG. 4, maps are shown of the WT1 protein. FIG. 4C defines the localization of all previously reported antigenic epitopes presented by HLA class I and II alleles; FIG. 4D depicts the location of immunogenic peptides identified in this report. As can be seen, the 11 epitopes previously reported to be presented by class I and 10 presented by class II HLA alleles are principally clustered in sequences encoded by exons 1, 7 and 10, while the epitopes recognized by normal T-cells sensitized with the WT1 peptide pool are principally clustered in sequences encoded by the first 5 exons. Thus, 26 of the new epitopes are included in each of the four major isoforms of WT1 resulting from splice variants that do or do not include the 17 amino acid sequence (aas 250-266) in exon 5 or the three amino acid sequence (400-410KTS) between zinc fingers 3 and 4. While the epitopes are broadly distributed, clusters of epitopes were detected in the RNA recognition domain in exon 1 and the activation domain (aa 181-250) (FIG. 4F) proximal to the spliced 17aa segment in exon 5. The latter area also contained those epitopes most frequently recognized by multiple donors (FIG. 4E). Interestingly, 9 newly identified epitopes map to a 126 amino acid sequence at the N terminus encoded by a segment of the WT1 gene initially described by Gessler et al (37) that is centromeric to exon 1 of the (Exon 5+, KTS+) isoform of WT1 and includes the long isoform of WT1 initiated at a CUG codon upstream of the AUG initiator for exon 1.50 Strikingly, each of the epitopes identified in this sequence elicits IFNγ+ T-cells that are cytolytic against leukemic blasts coexpressing WT1 and the T-cells' restricting HLA allele.
  • Of the several “self” proteins such as WT1, NY-ESO-1, HER2/neu, MAGE, and others, differentially expressed by specific tumors, only WT1 and MART-1 have been shown to elicit responses in normal donors (31,32,51-54). In contrast, T-cells specific for each of these proteins have been recorded in a proportion of patients with tumors overexpressing them (55). In particular, T-cells specific for the RMF and CMT peptides of WT1 have been detected in patients with leukemias, myeloma, carcinoma of the breast and prostate and other solid tumors (31,32,56-61). Responses to several of the WT1 epitopes identified in the present study in 50-60% of patients with ovarian cancer have been documented. Given the high number of potentially immunogenic epitopes in proteins such as NY-ESO-1 and HER2/neu that have elicited responses in tumor-bearing hosts (62), the number of immunogenic WT1 peptides we have identified is not sufficiently different to account for the differential presence of WT1 responses in normal donors. Furthermore, Pospori et al (63) have shown that HSCs expressing a transduced TCR specific for a WT1 peptide presented by HLA-A0201 are not deleted in the thymus of HLA-A0201 transgenic mice and generate functional memory T-cells. However, while the basis for this lack of “self” tolerance is unclear, the studies of Rezvani et al (31) and data herein (FIG. 1A) indicate that the frequencies of WT1 specific T-cells in the blood of healthy donors is low. In part, this may reflect the low levels and limited tissue distribution of WT1 expression in normal individuals (18-20). Recently, Rezvani et al (64) also demonstrated declining T-cell responses to WT1 in patients repeatedly vaccinated with WT1 peptides, suggesting that these responses are highly regulated. Lehe et al (65) have also recently shown that sensitization of T-cells with a WT1 peptide presented by DRB10402 in the presence of high concentrations of IL-2 preferentially stimulates the generation of CD25+ FOX P3+ GITR+CD127-regulatory T-cells capable of inhibiting CD8+WT1 specific T-cell responses.
  • Under the culture conditions employed herein, autologous DCs and EBVBLCL loaded with the WT1 peptide pool preferentially induced the generation of CD8+ and CD4+ IFNγ+WT1 peptide-specific T-cells from 41/56 normal donors (73%). Although each donor recognized only 1-3 epitopes of WT1, the fact that T-cells specific for 80% of these epitopes could recognize WT1+ leukemic cells sharing the T-cells' presenting HLA allele suggests that the turnover and processing of the aberrantly expressed WT1 is high, permitting the simultaneous presentation of several different WT1 epitopes by the restricting HLA allele expressed by these leukemic cells. Identification of these epitopes is useful both for in vitro generation of potent tumoricidal WT1 specific T-cells for adoptive cell therapies and for the generation of more broadly applicable vaccines for stimulating T-cell responses for eradication of clonogenic tumor cells expressing WT1 in vivo.
  • In one embodiment, peptides from the WT1 protein sequence that are upstream from exon 1, i.e., within the first 126 amino acids of SEQ ID NO:194, are heretofore unrecognized sites of immunogenic epitopes and therefore peptides useful for the purposes herein.
    • Example 1 Binding of HLA-A0201 and -A0301 by Synthetic Peptide Analogues Derived from WT1
  • Materials and Experimental Methods.
  • Peptides were synthesized by Genemed Synthesis Inc, CA using fluorenylmethoxycarbonyl chemistry and solid phase synthesis, and were purified by high pressure liquid chromatography (HPLC). The quality of the peptides was assessed by HPLC analysis, and the expected molecular weight was measured using matrix-assisted laser desorption mass spectrometry. Peptides were sterile and >90% pure. The peptides were dissolved in DMSO and diluted in PBS at pH 7.4 or saline solution to yield a concentration of 5 milligrams per milliliter (mg/ml) and were stored at −80° C. For in vitro experiments, an irrelevant control peptide, HLA A24 consensus, was used.
  • Peptide Sequence Analysis.
  • Peptide sequence analysis was performed using 2 databases. The first was the software of the Bioinformatics & Molecular Analysis Section (National Institutes of Health, Washington, D.C.) (Parker K C et al, Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol 152: 163-175, 1994), which ranks 9-mer or 10-mer peptides on a predicted half-time dissociation coefficient from HLA class I molecules. The second database, SYFPEITHI prediction software, is described in Rammensee H G et al (SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50: 213-219, 1999). Irrelevant control peptides used in in vitro experiments were: RAS (TEYKLVVVGAPGVGKSALTIQ; SEQ ID No: 198) or CML b2a2 (VHSIPLTINKEEALQRPVASDFE; SEQ ID No: 199) for Class II, and HIV pol (ILKEPVHGV; SEQ ID No: 200) or CML F (YLKALQRPY; SEQ ID No: 201) for Class I.
  • Cell Lines.
  • Cell lines were cultured in RPMI 1640 medium supplemented with 5% FCS, penicillin, streptomycin, 2 mM glutamine and 2-mercaptoethanol at 37° C. in humidified air containing 5% CO2. T2 is a human cell line lacking TAP1 and TAP2 and therefore unable to present peptides derived from cytosolic proteins. Raji cells are a human Burkitt lymphoma cells that exhibit a high level of TAP expression.
  • Human mesothelioma cell lines studied included: sarcomatoid (VAMT, H2373, H28), epithelioid (H2452) and biphasic (JMN, MSTO and H-Meso1A). Cell lines were obtained from the following sources: H-Meso1A: NCI, Bethesda, Md.; JMN and VAMT: Dr. Sirotnak, Memorial Sloan Kettering Cancer Center (MSKCC); H-2452 and H2373: Dr. Pass, Karmanos Cancer Institute, Wayne State University, Detroit, Mich.; H28 and MSTO: American Type Culture Collection (ATCC, Manassas, Va.). Cell lines were maintained in media recommended by the suppliers and incubated in a humidified incubator with 5% CO2.
  • Mesothelioma cell lines Meso 11, Meso 34, Meso 37, Meso 47 and Meso 56 were obtained from Dr. M Gregoire (Institute of Biology, Nantes, France) and cultured in RPMI 1640 (Life Technologies)+10% fetal calf serum (FCS), 1% penicillin—streptomycin, and 1% L-glutamine. All cells were HLA typed by the Department of Cellular Immunology at MSKCC. Melanoma cell line Mewo (WT1− A201+) was obtained from the ATCC. SKRC-52 renal cell carcinoma was obtained from L. Old of the Ludwig Institute. Leukemia cell lines were cultured in RPMI 1640+10% FCS, 1% penicillin-streptomycin, 2 mM glutamine and 2-mercaptoethanol at 37° C./5% CO2. LAMA81, BV173 and 697, Ph+ leukemias that are all WT1+ and A0201+, were provided by Dr. HJ Stauss (University College London). SKLY-16 is a human B cell lymphoma (WT1−, A0201+); K562, RwLeu4 and HL60, all WT1+ leukemias, were obtained from the ATCC.
  • T2 assay for peptide binding and stabilization of HLA A0201 molecules. T2 cells (TAP−, HLA-A0201+) were incubated overnight at 27° C. at a concentration of 1×106 cells/ml in FCS-free RPMI medium supplemented with 5 μg/ml human β2m (Sigma, St Louis, Mo.) in the absence (negative control) or presence of either a positive reference tyrosinase peptide or test peptides at various final concentrations (50, 10, 1, and 0.1 micrograms (μg)/ml). Following a 4-hour incubation with 5 μg/ml brefeldin A (Sigma), T2 cells were labeled for 30 minutes at 4° C. with a saturating concentration of anti-HLA-A2.1 (BB7.2) mAb, then washed twice. Cells were then incubated for 30 minutes, 4° C. with a saturating concentration of FITC-conjugated goat IgG F(ab′)2 anti-mouse Ig (Caltag, San Francisco, Calif.), washed twice, fixed in PBS/1% paraformaldehyde and analyzed using a FACS Calibur® cytofluorometer (Becton Dickinson, Immunocytometry Systems, San Jose, Calif.).
  • The mean intensity of fluorescence (MIF) observed for each peptide concentration (after dividing by the MIF in the absence of peptide) was used as an indication of peptide binding and expressed as a “fluorescence index.” Stabilization assays were performed similarly. Following initial evaluation of peptide binding at time 0, cells were washed in RPMI complete medium to remove free peptides and incubated in the continuous presence of 0.5 μg/ml brefeldin-A for 2, 4, 6 or 8 hours.
  • The number of stable peptide-HLA-A2.1 complexes was estimated as described above by immunofluorescence. The half time of complexes is an estimate of the time required for a 50% reduction of the MIF value at time=0.
  • WT1 Peptides.
  • The sequence of the WT1 protein published by Gessler et al. (37) which comprises 575 aminoacids and includes the first 126 aminoacids in the N-terminus missing in the (Exon 5+, KTS+) isoform of WT116, was used to design the peptide sequences (SEQ ID NO:194; FIG. 2A). 141 pentadecapeptides spanning this sequence, each overlapping the next by 11aa, were synthesized by Invitrogen (Baltimore, Md.) to specifications of validated sequence, 95% purity, sterility and absence of endotoxin. These 141 15-mers were mixed in equal amounts to form a total pool of peptides, in which each peptide is at a concentration of 0.35 mcg/ml. This pool was used for the T-cell sensitization. To identify peptides eliciting responses, subpools containing 12 pentadecapeptides (4.17 mcg/ml/peptide) were established to form a mapping matrix in which each peptide is included in only two overlapping subpools (FIG. 2B).
  • Generation of WT1 Specific T-Cells:
  • Peripheral blood was obtained from 56 consenting normal donors according to protocols approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center (New York, N.Y.). All donors were typed for HLA-A, B, C, DR and DQ at high resolution by standard techniques.
  • Cytokine-activated monocytes (CAMs) were used as antigen presenting cells (APCs), and generated as previously described (32). Briefly, peripheral blood monocytes were separated by adherence on plastic and cultured in RPMI1640 containing 1% autologous serum. GM-CSF (Berlex, Montville, N.J.) and interleukin-4 (IL-4) (R&D Systems, Minneapolis, Minn.) were added to final concentrations of 2000 U/ml and 1000 U/ml respectively on days 0, 2, 4. On day 5, these cells were additionally treated with TNFα(10 ng/ml), interleukin-6 (IL-6) (1000 IU/ml), (400 IU/ml), PGE2 (25 mM-3) (R&D Systems, Minneapolis, Minn.) together with GM-CSF and IL-4 at the same doses. CAMs harvested on day 7 of culture expressed CD83, CD80, CD86, and HLA class I and II alleles as determined by FACS analysis.
  • EBV-BLCL were also used as WT1 peptide loaded and control APCs or as targets as specified in the experiments. They were generated by infection of peripheral blood mononuclear cells (PBMC) with EBV strain B95.8 (38,39) as previously described. The EBV transformed BLCL (EBV-BLCL) were cultured in RPMI1640(Gemini) with 10% fetal calf serum (Gemini) in the presence of Acyclovir.
  • Sensitization and Propagation of WT1 Specific T-Cells.
  • To generate WT1-specific CTLs, PBMC were isolated by Ficoll-Hypaque density gradient centrifugation. Monocytes were depleted by adherence on plastic and NK cells by absorption to immunomagnetic CD56 pre-coated microbeads (Miltenyi Biotech Inc, MA) as previously described (32). Enriched T-cell fractions were stimulated at a 20:1 responder:stimulator ratio with autologous CAMs or EBV-BLCL that had been pre-loaded for 3 hours with the total pool of the WT1 pentadecapeptides in serum-free medium and irradiated to 3000 cGy. T-cells were cultured in Yssel's medium supplemented with 5% AB human serum (YHS, Gemini), re-stimulated weekly with the autologous WT1 total pool-loaded CAMs or EBV-BLCL and fed with interleukin-2(IL-2) (Collaborative Biomedical Products, Bedford, Mass.) every 2-3 days at 10-50 U/ml.
  • Cell Targets—Leukemic Cells:
  • Twenty-four primary leukemic cells and 1 leukemic cell line were characterized for their expression of WT1 by intracellular FACS staining using murine anti-human WT1 monoclonal antibodies (Neomarkers, Fremont, Calif.) as previously described (32,38) The WT1+ leukemias included blast cells from 11 primary AMLs, 3 primary ALLs and 1 B-cell precursor ALL cell line. Ten WT1− leukemias, were used as controls, and included 3 B-cell precursor ALLs and 7 AMLs.
  • All EBV BLCL and leukemia cells were typed for HLA A, B, C, DR and DQ alleles at high resolution by standard techniques.
    • Assessment of T-Cell Response.
  • IFNγ Production by WT1 Specific T-Cells.
  • The proportion and phenotype (CD4 and CD8) of T-cells generating IFNγ in response to secondary stimulation with the WT1 total pool, WT1 subpools or single WT1 15-mer or 9-mer WT1 peptides loaded on autologous PBMC were measured by FACS analysis of T-cells containing intracellular IFNγ as previously described (38,40).
  • Mapping of Immunogenic Epitopes.
  • Aliquots of the T-cells stimulated with the WT1 total pool for 35-42 days were washed and re-stimulated overnight with autologous PBMC loaded with one of each of the subpools of WT1 pentadecapeptides. T-cell responses to each subpool were quantitated by FACS analysis of T-cells bearing intracellular IFNγ as previously described (41). The mapping grid (FIG. 2B) was then used to identify specific WT1 15-mers uniquely shared by 2 subpools eliciting T-cell responses. These 15-mers and 9-mer or 11-mer sequences within the 15-mers were then analyzed as secondary single peptide stimulators to confirm their immunogenicity and define the immunogenic epitope(s) within the 15-mer eliciting responses.
  • Cytotoxic Activity.
  • The W-1-specific and HLA-restricted cytotoxic activity of sensitized T-cells was measured in standard Cr51 release assays against a panel of HLA-matched and mismatched CAM targets either unmodified or loaded with the total pool, the identified 15-mer, or the 9-mer or 11-mer epitope of WT1 eliciting T-cell responses, as previously described (32). In addition, the restricting HLA allele presenting each immunogenic epitope was identified by measuring the cytotoxicity of the sensitized T-cells against a panel of allogeneic CAMs pre-loaded with the peptide, each sharing a single HLA allele expressed on the responding WT1-specific T-cells as previously described (41). The cytotoxic activity of the WT1 epitope-specific CTLs against WT1− and WT1+ leukemia cell lines or primary leukemic cells expressing the restricting HLA alleles was also assessed in this cytotoxicity assay Cr51 assay as previously described (32).
  • Immunogenicity of the Identified Immunodominant WT1 Derived Epitopes.
  • To estimate the immunogenicity of identified WT1 peptide epitopes in different individuals, enriched T-cells separated from PBMC of groups of normal donors expressing one of a series of prevalent HLA alleles (i.e. HLA-A0201, A0301, A2402, B0702) which were previously identified as a presenter of a newly identified WT1 epitope were sensitized in vitro with artificial antigen-presenting cells (AAPC) (42) expressing that HLA allele and loaded with the pre-identified WT1 epitope or an irrelevant peptide. The panel of AAPCs includes AAPCs expressing one of the following single HLA alleles: HLA A0201, A0101, A0301, A2402, B0702 or B0801, which were generated as previously described (42). After 35 days of co-culture of T-cells with the peptide-loaded AAPCs in the presence of IL2, CTLs were secondarily stimulated overnight with autologous PBMC loaded with the sensitizing peptide or an unrelated peptide and tested for their IFNγ response. The responses were registered as positive if the proportion of T-cells producing IFNγ in response to the secondary stimulation with autologous PBMC loaded with the stimulating WT1 derived peptide exceeded the background proportion of IFNγ T-cells incubated with PBMC alone by two fold or more.
    • Example 2 Responses of Normal Donors to the WT1 Total Pool of Pentadecapeptides
  • Frequencies of WT1-specific IFNγ+ T-cells in the PBMC of 41 normal donors were measured initially. These frequencies ranged between 0.01% to 1.82%, and exceeded the background of IFNγ+ T-cells detected in T-cells stimulated with autologous PBMC alone in only 10/41 individuals (FIG. 1A). In vitro sensitization of T-cells from 56 normal donors with autologous CAMs loaded with the total pool of WT1 pentadecapeptides for periods of 35-42 days resulted in significant expansion of IFNγ+ T-cells in 41/56 cases (73%) (FIG. 1A). T-cells generated from 38/56 donors also exhibited cytotoxic activity against autologous PHA blasts loaded with the WT1 total pool (FIG. 1B), including T-cells from 38 of the 41 donors that produced IFNγ in response to secondary stimulation with the WT1 peptide pool.
  • The capacity of one of the previously reported WT1 epitopes predicted to bind the HLA-A0201 allele, 126-134RMFPNAPYL (SEQ ID NO:161; RMF) (43) were compared with the total pool of WT1 pentadecapeptides to stimulate WT1 reactive T-cells in HLA-A0201+ normal donors (n=14) when loaded on autologous CAMs. Increased frequencies of IFNγ+ T-cells initially sensitized with the RMF peptide were detected in 9/14 donors, 7 of whom also responded to secondary simulation with the pooled peptides (FIG. 1C). In contrast, 12/14 CTL lines initially sensitized with the WT1 peptide pool, generated high frequencies of IFNγ+ T-cells after secondary stimulation with the WT1 total pool, including 6 CTL lines that also responded to RMF. The epitopes of WT1 recognized by the T-cells sensitized with the total pool (vide infra) were mapped and epitopes other than RMF in 12/14 donors were identified. The magnitude of the responses to those epitopes was much higher than to the RMF peptide (FIG. 1C). Only 4/14 CTL lines initially sensitized with RMF exhibited cytotoxic activity against RMF-loaded autologous PHA blasts; of which 3 could also lyse autologous PHA blasts loaded with the WT1pool (FIG. 1D). In contrast, 10/14 CTL sensitized with the pool of WT1 peptides were cytotoxic against PHA blasts loaded with the WT1 total pool including 3/14 that lysed RMF peptide loaded blasts (FIG. 1D). Thus, in a high proportion of HLA A0201+ donors, stimulation of T-cells with the WT1 total pool more consistently elicited WT1-specific T-cell responses than stimulation with the single HLA A0201 binding RMF peptide.
  • Detailed description of FIG. 1. WT1 specific responses of CTL generated from PBMC of normal donors (n=56) by stimulation with autologous APCs loaded with total pool of WT1 derived pentadecapeptides: A. production of IFNγ in PBMC alone (as a background), PBMC co-incubated overnight with the total pool of pentadecapeptides spanning the whole sequence of WT1 protein (PBMC+WT1 pool) and pre-generated WT1 specific T cells co-incubated overnight with WT1 peptide loaded PBMC; B. cytotoxic activity of the WT1 specific CTLs generated in vitro by stimulation with WT1 total pool against WT1− (autologous PHA stimulated blasts) and WT1+(autologous PHA stimulated blasts loaded with the total pool of WT1 pentadecapeptides) targets at 50:1 effector:stimulator ratio; C. IFNγ response measured by FACS staining in different responder cell populations (peripheral blood derived PBMC, pre-generated CTLs sensitized in vitro with the RMF peptide loaded on autologous CAM and pre-generated CTLs sensitized with the total pool of WT1 15-mers) after secondary overnight stimulation with autologous PBMC either unmodified or loaded with one of the following: RMF peptide, dominant epitopes of WT1 identified by the epitope mapping approach in the WT1-total pool sensitized CTL, WT1 total pool of the 141 pentadecapeptides; D. Cytotoxic activity of the WT specific T cells generated in vitro by sensitization with autologous CAMs loaded with the RMF 9-mer or with the total pool of the WT1 15-mers. The cytotoxicity of the T cells was assessed against autologous WT1 negative targets(PHA activated blasts) and the same targets loaded with RMF peptide, the total pool of WT1 15-mers or the dominant WT1 epitope identified for the same T cell line.
    • Example 3 Identification of Immunogenic Epitopes of WT1 Protein Recognized by the WT1-Reactive T-Cells
  • WT1 CTLs Generated by Sensitization with the Pooled Peptides are Epitope Specific and HLA Restricted.
  • The epitopes recognized by T-cells sensitized in vitro with the total pool of overlapping WT1 pentadecapeptides (FIG. 2A) were identified by quantitating IFNγ+ T-cells responding to a mapping grid of subpools of WT1 15-mers formed so that any single 15-mer is shared by only 2 intersecting subpools (FIG. 2B). As shown for a representative example in FIG. 2C, significantly increased numbers of IFNγ+ T-cells are selectively generated in response to subpools #3 and #19 which share the pentadecapeptide #75. The T-cells were then stimulated with neighboring 15-mers, each overlapping peptide #75 by 11aa. As can be seen, IFNγ+ T-cells are selectively generated in response to peptide #75 (FIG. 2D). The newly identified immunogenic WT1 epitope is 174-182 HSFKHEDPM. Subsequently, the cytotoxic activity of these T-cells was assessed against a panel of allogeneic CAMs either unmodified or loaded with this peptide, each sharing one HLA allele expressed by the tested CTLs. As shown in FIG. 2E, the T-cells selectively lysed peptide loaded autologous targets and targets expressing the HLA-B3501 allele, and did not lyse peptide-loaded targets sharing other HLA alleles inherited by the T-cell donor. These T-cells also lysed WT1+ BALL cells coexpressing the HLA-B3501 allele.
  • Detailed description of FIG. 2. Strategy for the generation of the total pool of overlapping pentadecapeptides spanning the whole sequence of the WT1 protein and epitope mapping: A. The sequence of the WT1 protein consisting of 575 amino acids and the principle of 11 amino acid overlapping pentadecapeptides are illustrated. A total of 141 pentadecapeptides are required to span the entire protein. The sequence of 575 aminoacids published by Gessler et al. (37), was employed. This sequence includes an additional 126 aminoacids in the N-terminus. In order to match the sequential numbers of aminoacids within the WT1 sequence used with the longest, most frequently described WT1 isoform D we numbered the first 126aa with negative values and used the positive values to number the subsequent 449 aminoacids described in the longest isoform D; B. The mapping grid consisting of 24 subpools each containing up to 12 WT1-derived pentadecapeptides. Each peptide is uniquely contained within two intersecting subpools: for example peptide 75 is uniquely shared by subpools 3 and 19; C. IFNγ production by WT1 sensitized CTLs in response to secondary overnight stimulation with the subpools of WT1 pentadecapeptides loaded on autologous PBMC. Dominant responses are observed for the subpools #3 and #19 both containing one common pentadecapeptide #75; D. IFNγ production by the WT1 CTLs in response to secondary overnight stimulation with the single pentadecapeptide contained within the subpools eliciting the highest responses as per the analysis determined in 2C of this figure confirms that the dominant immunogenic sequence is contained within pentadecapeptide #75; E. HLA restriction of the WT1 specific T cells responding to peptide #75 identified by Cr51 release assay against a panel of allogeneic CAMs or PHA blasts matching single HLA alleles expressed by the WT1 CTL donors. These are presented along the X axis of the graph. The CAMs or PHA blasts used in the assay are unmodified (grey bars) or loaded with the WT1 dominant epitope (black bars). The WT1 specific cytotoxic activity of the WT1 CTLs is restricted by the B3501 HLA allele.
  • Mapping of WT1 Peptides Eliciting T-Cell Responses Identifies a Diversity of Immunogenic Epitopes Presented by Different Class I and II HLA Alleles.
  • The same approach was used to map and ultimately identify WT1 epitopes eliciting responses by T-cells from the other 40 responding normal donors. Of these donors, 8 (19%) responded exclusively to one WT1 peptide, while 18 (43%) responded to two and 16 (39%) to 3 peptides. In cultures eliciting responses to more than one WT-1 peptide, the patterns of IFNγ+ T-cell responses to the subpools were sufficiently distinctive to permit initial segregation of potentially immunogenic peptides. Each candidate peptide was then evaluated individually to ascertain the specific peptide inducing a T-cell response.
  • The immunogenic peptides of WT1 that were identified and their presenting HLA alleles are listed in Table 1. Of the 42 WT1 peptides eliciting T-cell responses, 41 are newly identified; only one of these WT1 peptides, the 126-134 RMFPNAPYL nonamer presented by HLA-A0201, has been previously described and shown to be immunogenic when presented by this allele (43) Peptide 91, 235-249CMTWNQMNLGATLKG contains an epitope which, in the study, elicited CD4+ T-cell responses restricted by HLA DRB1 0402, but also contains the 235-243CMT nonamer known to be presented by HLA A0201 and HLA A2402 (29). For 26 of the peptides presented by class I HLA alleles, a single presenting HLA allele was identified in the initially studied donor. However, when the HLA-restrictions of T-cells responding to these peptides in different donors was examined, 10 of these peptides were found that could elicit T-cell responses when presented by 2 or 3 different class I HLA alleles. One sequence, the 238-246WNQMNLGAT peptide, elicited strong IFNγ+CD8+ T-cell responses when presented in different donors by any one of 4 distinct HLA class I alleles.
  • TABLE 1
    WT1 derived immunogenic epitopes identified by IFNγ production
    assay for T cells responses using pool of overlapping pentadecapeptides spanning the whole
    sequence of WT1protein. Bolded peptide sequences represent those tested in Example 5 and
    results shown in Table 3.
    Cytotoxic CTL response,
    15-mer % (at 50:1) E:T ratio vs
    number IFNg response of WT1
    (SEQ ID, cells, % IFNg + peptide
    Table IV) cells WT1- loaded
    Containing the Present- WT1 Auto- auto- WT1 − WT1 +
    dominant ing HLA No WT1 peptide logous logous leu- leu-
    epitope Sequence identified allele peptide loaded APC APC kemia kemia
    #1 (−125)-(−117) B0702 0.9 11.3 0 27 1 67
    RQRPHPGAL
    (SEQ ID NO: 142)
    #2 (−119)-(−111) B0702 0.5 14.0 0 30 1 60
    GALRNPTAC
    (SEQ ID NO: 143)
    #4 (−110)-(−102) A0201 0.98 5.75 0 30 2 22
    PLPHFPPSL
    (SEQ ID NO: 144)
    #5 (−107)-(−99) A3101 0.73 4.82 0 42 ND ND
    HFPPSLPPT
    (SEQ ID NO: 145)
    #7 **** (−99)-(−91) B4001 1.5 12.8 0 45 3 65
    THSPTHPPR A0201 0.4 5 2 50 0 38
    (SEQ ID NO: 146)
    #13 (−75)-(−67) A0201 0.61 5.07 0 18 3 19
    AILDFLLLQ
    (SEQ ID NO: 147)
    #20 **** (−47)-(−39) A0201 0.2 3.67 6 54 5 19
    PGCLQQPEQ B4701 0.5 4.6 6 54 ND ND
    (SEQ ID NO: 148)
    (−47)-(−37) DRB10101 0.33 3.1 6 54 ND ND
    PGCLQQPEQQG
    (SEQ ID NO: 149)
    #24-25 (−27)-(−19) A0201 1.05 4.48 3 41 10 37
    KLGAAEASA
    (SEQ ID NO: 150)
    #29-30 (−8)-(1) B3501 0.07 1.0 5 73 5 39
    ASGSEPQQM
    (SEQ ID NO: 151)
    #33 **** 6-15 A0201 1.1 11.0 2 51 0 9
    RDLNALLPAV** B5701 0.19 1.24 3 44 ND ND
    (SEQ ID NO: 152)
    #37 22-31 A0201 0.07 0.9 8 32 3 47
    GGCALPVSGA
    (SEQ ID NO: 153)
    #39 30-38 B3901 0.1 1.3 2 31 ND ND
    GAAQWAPVL
    (SEQ ID NO: 154)
    #41 **** 38-46 A0201 0.2 4.18 0 73 0 40
    LDFAPPGAS
    (SEQ ID NO: 155)
    38-48 DRB10402 0.2 1.41 0 73 0 40
    LDFAPPGASAY
    (SEQ ID NO: 156)
    #43 **** 46-54 A0201 1.2 6.46 2 51 0 0
    SAYGSLGGP* B4001 1.09 6.84 2 41 3 68
    (SEQ ID NO: 157)*
    #46 58-66 A0201 1.15 6.69 2 40 0 0
    PAPPPPPPP**
    (SEQ ID NO: 158)
    #58 106-114 B4402 0.92 5.65 8 46 ND ND
    ACRYGPFGP
    (SEQ ID NO: 159)
    #62 **** 122-130 B3503 0.78 2.0 0 84 ND ND
    SGQARMFPN*** C0401 0.78 2.0 0 84 ND ND
    (SEQ ID NO: 160)
    #62-63 126-134 A0201 0.52 2.17 3 41 2 25
    RMFPNAPYL*
    (SEQ ID NO: 161)
    #65-66 135-143 B3501 0.07 0.61 0 35 ND ND
    PSCLESQPA
    (SEQ ID NO: 162)
    #68 146-154 A0101 0.92 4.0 2 19 ND ND
    NQGYSTVTF
    (SEQ ID NO: 163)
    #73 166-174 B3801 0.81 3.14 0 26 ND ND
    HHAAQFPNH
    (SEQ ID NO: 164)
    #74-75 174-182 B3501 1.3 18.0 0 50 5 45
    HSFKHEDPM
    (SEQ ID NO: 165)
    #82 202-210 B4402 1.02 3.77 8 37 ND ND
    CHTPTDSCT
    (SEQ ID NO: 166)
    #83-84 209-217 A0101 0.03 0.29 0 21 3 33
    CTGSQALLL
    (SEQ ID NO: 167)
    #83 **** 206-214 B3802 0.71 4.02 0 88 ND ND
    TDSCTGSQA B4402 1.01 4.2 1 36 1 56
    (SEQ ID NO: 168)
    #86 **** 218-226 B3503 0.84 3.0 0 84 4 48
    RTPYSSDNL*** C0401 0.84 3.0 0 84 4 48
    (SEQ ID NO: 169)
    #87 225-233 A0201 0.13 0.9 3 87 0 0
    NLYQMTSQLE**
    (SEQ ID NO: 170)
    #91 **** 238-246 A0201 1.34 8.0 0 18 1 19
    WNQMNLGAT C1701 2.1 12.0 0 10 1 16
    (SEQ ID NO: 171) A0101 2.1 7.31 0 26 ND ND
    B3508 1.23 5.0 0 18 4 19
    #91-92 239-248 A2402 0.02 0.14 4 9 1 17
    NQMNLGATL
    (SEQ ID NO: 172)
    #91 **** 238-248 DRB11104 0.59 6.0 0 8 0 0
    WNQMNLGATLK
    (SEQ ID NO: 173)
    235-249 DRB10402 0.07 0.53 4 16 1 17
    CMTWNQMNLGAT
    LKG
    (SEQ ID NO: 174)
    #92 **** 242-250 A0101 0.32 1.83 2 19 ND ND
    NLGATLKGV A0201 0.06 0.75 1 18 2 19
    (SEQ ID NO: 175)
    #92-93 243-252 A0203 0.54 2.1 0 35 ND ND
    LGATLKGVAA
    (SEQ ID NO: 176)
    #93 246-253 A6901 0.09 1.85 4 80 ND ND
    TLGVAAGS
    (SEQ ID NO: 177)
    #99-100 **** 269-278 A0101 0.12 2.43 0 27 0 33
    GYESDNHTT B3501 0.1 0.61 0 35 ND ND
    (SEQ ID NO: 178)
    #112-113 **** 323-332 B3501 1.3 18.0 0 70 5 45
    FMCAYPGCNK
    (SEQ ID NO: 179)
    320-334 DRB10401 0.91 3.48 9 5 5 5
    KRPFMCAYPGC
    (SEQ ID NO: 180)
    #129 390-398 A0201 1.08 5.81 3 40 ND ND
    RKFSRSDHL
    (SEQ ID NO: 181)
    #131 398-406 A0201 1.56 14.0 0 38 ND ND
    LKTHTRTHT
    (SEQ ID NO: 182)
    #141 **** 436-445 A0201 1.78 6.69 2 40 0 0
    NMHQRNHTKL** B4001 2.1 7.71 0 31 3 72
    (SEQ ID NO: 183) A2402 0.61 2.79 19 47 0 0
    * - the epitope previously predicted by the computer algorithm or described in the literature
    ** - T-cells cytotoxic against autologous WT1 peptide loaded APCs but not leukemia cells
    *** - assignment of HLA restriction to one or other allele cannot be made due to lack of targets inheriting one allele without the other
    **** - rows represent peptides that can be presented by more than one HLA allele.
  • Using this epitope mapping strategy, 5 new 11-mer peptides were identified that stimulated CD4+ T-cell responses restricted by HLA class II alleles. The CD4+ T-cells generated in response to each of these epitopes expressed high levels of IFNγ+ T-cells. The CD4+ T-cells responding to 3 of these 5 peptide epitopes also exhibited specific cytotoxic activity against peptide loaded PHA blasts as well as unmodified WT1+ leukemic blasts selectively sharing the restricting class II HLA allele.
  • In 4 of the 56 donors tested, epitope mapping of T-cells sensitized with the complete pool of WT1 15-mers identified specific 15-mers eliciting both CD4+ and CD8+ T-cell responses (15- mer peptides # 20, 41, 91, 112). Fine mapping of the sequences eliciting these responses identified four 11-mers that stimulated HLA class II-restricted CD4+ T-cell responses which also contained, within their sequences, 9-mers that elicited HLA class I-restricted CD8+ T-cell responses. A representative example of one of these dual stimulating peptides is presented in FIG. 3. In this case, peptide 41 was found to elicit both CD4+ and CD8+ IFNγ+ T-cell responses (FIG. 3A). Fine mapping of the 11-mers within peptide 41 eliciting the CD4+ IFNγ+ T-cell response (FIG. 3A) suggested the 38-48LDFAPPGASAY peptide as the most immunogenic sequence inducing both CD4+ and CD8+ IFNγ+ T-cell responses. Strikingly, the peptide 41 sensitized T-cells lysed PHA blasts sensitized with either the 9aa sequence (38-46LDFAPPGAS) or the 11aa sequence (38-48LDFAPPGASAY), but did not lyse PHA blasts loaded with the 36-46PVLDFAPPGAS or 37-47VLDFAPPGASA 11-mers. Subsequent examination of the HLA restriction of the T-cells in the culture (FIG. 3D) revealed that the class II HLA-restricted T-cells were selectively cytotoxic against targets sharing the alleles DRB1 0402 and DQB1 0302 only when loaded with the LDF 11-mer, while the T-cells restricted by HLA A0201 were able to lyse targets loaded with either the 11-mer or the 9-mer LDF peptide. In this case, it was not possible to ascertain whether DRB1 0402 or DQB1 0302 was the restricting class II HLA allele because cells were not available in the panel expressing one without the other.
  • Detailed description of FIG. 3. HLA class I and II restricted WT1 specific T cell respond to the same immunodominant peptide 15-mer derived from WT1 protein in the WT1 CTL sensitized with the WT1 total pool of overlapping 15-mers loaded on autologous CAMs. A. Production of IFNg by the CD8+ and CD4+WT1 specific T cells in response to secondary overnight stimulation with the same dominant WT1 derived 15-mer #41; B. Identification of the immunogenic sequence of aminoacids within pentadecapeptide #41 by IFNg production after secondary overnight stimulation with autologous PBMC loaded with a panel of 9-mers either unique for the peptide #41 (LDF—LDFAAPGAS) or contained within the neighboring overlapping 15-mer #40 (PVL—PVLDFAPPG, VLD—VLDFAPPGA) and #42 (DFA—DFAPPGASA). Only the 9-mer uniquely presented within the 15-mer #41, LDF, elicits an IFNg response; C. Peptide-specific cytotoxic activity of WT1 CTL against the panel of 9-mers and 11-mers contained within peptide #41 and loaded on autologous PHA stimulated blasts is observed against both the 11-mer LDF and 9-mer LDF contained within the 11-mer LDF as determined in a standard Cr51 release assay at 25:1 E:T ratio; D. HLA restriction of the cytotoxic activity of the WT1 CTL: T-cells restricted by HLA-A0201 lyse targets loaded with either the 11-mer or the 9-mer, while those restricted by HLA DRB10402 only lysed targets loaded with the 11-mer.
    • Example 4 T-Cells Generated Against Newly Identified WT1 Epitopes Exhibit Cytotoxic Activity Against WT1+ Leukemias
  • Once the WT1 peptide specificity was established and HLA restrictions of the IFNγ+ T-cells responding to the pool of WT1 peptides, their cytotoxic activity was examined against unmodified and peptide loaded autologous PHA blasts and against a series of allogeneic PHA blasts loaded with the identified peptides as well as primary acute leukemic cell blasts expressing WT1 protein that coexpressed the WT1 specific T-cells' restricting HLA allele. For the latter tests, WT1+ leukemic cells not expressing the restricting allele and WT1− cells sharing the restricting allele served as controls. Results are summarized in Tables 1 and 2.
  • As can be seen in Table 1, of 51 cultures generating IFNγ+CD8+ T-cells after secondary stimulation with an identified peptide loaded autologous APC, 50 also exhibited significant specific cytotoxic activity against autologous PHA blasts loaded with the targeted peptide. Of these, 48 also lysed allogeneic peptide loaded PHA blasts or DCs sharing the restricting HLA allele of the responding T-cells. CD4+ IFNγ+ T-cells responding to ⅗ identified 11-mer peptides presented by class II HLA alleles also lysed peptide loaded autologous and HLA-sharing allogeneic class II+ targets.
  • Of the T-cell cultures exhibiting epitope-specific cytotoxic activity against peptide loaded targets, 36 could be tested for cytotoxic activity against WT1+ leukemic cells coexpressing the T-cell's restricting HLA allele. Of these 36, 27 exhibited HLA-restricted cytotoxic activity against the WT1+ leukemic cells (Table 2). T-cells specific for five peptides, 6-15RDL, 46-54SAY, 58-66PAP, 225-233NLY, and 436-445NMH, presented by HLA A0201, could not lyse HLA-A0201+ WT1+ leukemic cells. However, HLA B4001 restricted T-cells specific for the 46-54SAY peptide, could lyse WT1+ leukemic coexpressing this HLA allele. Similarly, NMH peptide-specific HLA-restricted T-cell lines that lysed targets loaded with the NMH peptide coexpressing HLA A0201, B4001 or A2402 were only able to lyse WT1+ leukemic cells expressing the HLA B4001 allele.
  • TABLE 2
    WT1 derived immunogenic epitopes identified by IFNγ production
    assay for T cells responses using pool of overlapping pentadecapeptides spanning the whole
    sequence of WT1 protein. Bold sequences indicate peptides tested as described in Example 5
    and results provided in Table 3.
    Cytotoxic CTL response, % (at 50:1)E:T ratio vs
    Prediction WT1- WT1 + allo
    algorithm allo APC APC with
    Dis- with restricting
    associa- restrict- HLA allele
    Presenting Binding tion ing HLA loaded with WT1− WT1+
    HLA allele Sequence identified index time allele WT1 peptide leukemia leukemia
    A0101 146-154 3 0.001 4 15 ND ND
    NQGYSTVTF
    SEQ ID NO: 163
    209-217 12 0.125 0 26 3 33
    CTGSQALLL
    SEQ ID NO: 167
    238-246 2 0 3 19 ND ND
    WNQMNLGAT
    SEQ ID NO: 171
    242-250 3 0.01 1 17 ND ND
    NLGATLKGV
    SEQ ID NO: 175
    269-278 15 1.5 0 26 0 33
    GYESDNHTT
    SEQ ID NO: 178
    323-332 0 0.1 2 0 5 0
    FMCAYPGCNK**
    SEQ ID NO: 179
    A0201 (−110)-(−102) 21 2 1 24 2 22
    PLPHFPPSL
    SEQ ID NO: 144
    (−99)-(−91) 3 0 1 21 0 38
    THSPTHPPR
    SEQ ID NO: 146
    (−75)-(−67) 19 0.272 3 17 3 19
    AILDFLLLQ
    SEQ ID NO: 147
    (−47)-(−39) 0 0 7 27 5 19
    PGCLQQPEQ
    SEQ ID NO: 148
    (−27)-(−19) 19 17 2 22 10 37
    KLGAAEASA
    SEQ ID NO: 150
    6-15 18 0.2 4 31 0 9
    RDLNALLPAV
    SEQ ID NO: 152
    22-31 13 0.003 3 25 3 47
    GGCALPVSGA
    SEQ ID NO: 153
    38-46 11 0 1 62 0 40
    LDFAPPGAS
    SEQ ID NO: 155
    46-54 14 0 5 31 0 0
    SAYGSLGGP**
    SEQ ID NO: 157
    58-66 5 0 1 18 0 0
    PAPPPPPPP**
    SEQ ID NO: 158
    126-134 22 313 1 52 2 25
    RMFPNAPYL*
    SEQ ID NO: 161
    225-233 23 68 3 28 0 0
    NLYQMTSQLE**
    SEQ ID NO: 170
    238-246 19 0.3 0 21 1 19
    WNQMNLGAT
    SEQ ID NO: 171
    242-250 24 160 1 14 2 19
    NLGATLKGV
    SEQ ID NO: 175
    390-398 11 0.054 1 27 ND ND
    RKFSRSDHL
    SEQ ID NO: 181
    398-406 5 0.18 1 22 ND ND
    LKTHTRTHT
    SEQ ID NO: 182
    436-445 20 15 4 32 0 0
    NMHQRNHTKL**
    SEQ ID NO: 183
    A0203 243-252 19 NA 0 21 ND ND
    LGATLKGVAA
    SEQ ID NO: 176
    A2402 239-248 10 7.2 0 2 1 17
    NQMNLGATL
    SEQ ID NO: 172
    436-445 13 0.6 13 27 0 0
    NMHQRNHTKL**
    SEQ ID NO: 183
    A6901 246-253 NA NA 0 57 ND ND
    TLGVAAGS
    SEQ ID NO: 177
    B0702 (−125)-(−117) 15 40 1 53 1 67
    RQRPHPGAL
    SEQ ID NO: 142
    (−119)-(−111) 2 0.3 5 22 1 60
    GALRNPTAC
    SEQ ID NO: 143
    A3101 (−107)-(−99) NA 0.01 0 27 ND ND
    HFPPSLPPT
    SEQ ID NO: 145
    B3501 (−8)-(−1) NA 15 3 51 5 39
    ASGSEPQQM
    SEQ ID NO: 151
    135-143 NA 0.075 0 21 ND ND
    PSCLESQPA
    SEQ ID NO: 162
    174-182 NA 10 3 63 5 45
    HSFKHEDPM
    SEQ ID NO: 165
    269-278 NA 0.004 0 23 ND ND
    GYESDNHTT
    SEQ ID NO: 178
    323-332 NA 0.01 0 61 5 45
    FMCAYPGCNK
    SEQ ID NO: 179
    B3503 122-130 NA NA 3 41 ND ND
    SGQARMFPN
    SEQ ID NO: 160
    218-226 NA NA 3 31 4 48
    RTPYSSDNL
    SEQ ID NO: 169
    B3508 238-246 NA NA 2 21 4 19
    WNQMNLGAT
    SEQ ID NO: 171
    B3802 206-214 NA NA 1 53 ND ND
    TDSCTGSQA
    SEQ ID NO: 168
    B3801 166-174 11 0.3 1 17 ND ND
    HHAAQFPNH
    SEQ ID NO: 164
    B3901 30-38 12 3 0 19 ND ND
    GAAQWAPVL
    SEQ ID NO: 154
    B4001 (−99)-(−91) 3 0.02 0 31 3 65
    THSPTHPPR
    SEQ ID NO: 146
    46-54 1 0.002 8 24 3 68
    SAYGSLGGP
    SEQ ID NO: 157
    436-445 1 0.002 1 26 3 72
    NMHQRNHTKL
    SEQ ID NO: 183
    B4402 202-210 3 NA 7 19 ND ND
    CHTPTDSCT
    SEQ ID NO: 166
    206-214 2 NA 0 88 1 56
    TDSCTGSQA
    SEQ ID NO: 168
    106-114 4 NA 7 23 ND ND
    ACRYGPFGP
    SEQ ID NO: 159
    B4701 (−47)-(−37) 1 NA 1 25 ND ND
    PGCLQQPEQ
    SEQ ID NO: 148
    B5701 6-15 NA NA 1 22 ND ND
    RDLNALLPAV
    SEQ ID NO: 152
    C0401 122-130 NA NA 3 41 ND ND
    SGQARMFPN
    SEQ ID NO: 160
    C1701 238-246 NA NA 0 7 1 16
    WNQMNLGAT
    SEQ ID NO: 171
    DRB10101 (−47)-(−37) 8 NA 1 25 ND ND
    PGCLQQPEQQG
    SEQ ID NO: 149
    DRB10402 38-48 NA NA 1 71 0 40
    LDFAPPGASAY
    SEQ ID NO: 156
    DRB10402 235-249 NA NA 2 15 1 17
    CMTWNQMNLGA
    TLKG
    SEQ ID NO: 174
    DRB10401 320-334 22 NA 3 0 5 5
    KRPFMCAYPGC
    SEQ ID NO: 180
    DRB11104 238-248 NA NA 2 1 0 0
    WNQMNLGATLK
    SEQ ID NO: 173
    * - previously reported epitopes;
    ** - T cells cytotoxic against the autologous WT1 peptide loaded APC but not the leukemic cells.
  • To ascertain that the cytotoxic activity of the WT1 peptide-specific T-cells observed against allogeneic WT1+ leukemic cells sharing the T-cells restricting allele does not reflect the presence of alloresponsive T-cells in the T-cell lines, we tested the cytotoxic activity of 13 of these HLA-restricted WT1 peptide specific T-cell lines against WT1+ leukemic cells and WT1− PHA blasts cultured from the same leukemic patient. As shown in Table 3a, the WT1 specific T-cells lysed the WT1+ leukemic cells but not PHA blasts from the same patient.
  • TABLE 3a
    Cytotoxic activity of the T cells specific for WT1 derived
    immunogenic epitopes identified by IFNγ production assay for T cells responses using pool of
    overlapping pentadecapeptides spanning the whole sequence of WT1 protein and tested against
    WT1 positive primary leukemic cells and PHA blasts of the same origin.
    Cytotoxic CTL
    response,
    15-mer % (at 50:1) E:T
    number ratio vs
    Containing Presenting WT1+ PHA
    the dominant HLA Leukemia blasts
    epitope Sequence identified allele ** ***
    #1 (−125)-(−117) RQRPHPGAL B0702 67 2
    SEQ ID NO: 142
    #2 (−119)-(−111) GALRNPTAC B0702 60 1
    SEQ ID NO: 143
    #4 (−110)-(−102) PLPHFPPSL A0201 22 1
    SEQ ID NO: 144
    #7 (−99)-(−91) THSPTHPPR B4001 65 5
    SEQ ID NO: 146 A0201 38 3
    #24-25 (−27)-(−19) KLGAAEASA A0201 37 8
    SEQ ID NO: 150
    #29-30 (−8)-(−1) ASGSEPQQM B3501 39 9
    SEQ ID NO: 151
    #37 22-31 GGCALPVSGA A0201 47 6
    SEQ ID NO: 153
    #43 46-54SAYGSLGGP* B4001 68 3
    SEQ ID NO: 157
    #62-63 126-134RMFPNAPYL* A0201 25 3
    SEQ ID NO: 161
    #86 218-226 RTPYSSDNL B3503 48 1
    SEQ ID NO: 169 C0401 48 1
    #141 436-445NMHQRNHTKL* B4001 72 1
    SEQ ID NO: 183
    P < 0.001
    * - the epitope previously predicted by the computer algorithm or described in the literature
    ** - leukemia samples were presented either by immortalized leukemia cell lines or by primary leukemia cells obtained from patients with WT1+ leukemia
    *** - PHA blasts were generated from PBMC derived from the same patients as the WT1+ primary leukemia
  • PHA blasts were not available from every patient that provided leukemia blasts for this study. Nevertheless, these results provide evidence that the cytotoxicity of the WT1 specific T-cells is not ascribable to contaminating alloreactivity. A second, more inclusive, but less direct line of evidence is provided by a paired comparison of the responses of T-cells derived from 35 of the donors that had been contemporaneously sensitized in vitro against either WT1 peptide pool loaded or unmodified autologous EBVBLCL, against these primary leukemias. As shown in Table 3b, T-cells sensitized with the WT1 peptide pool-loaded EBVBLCL lysed WT1+ leukemic cells sharing the T-cells' restricting HLA allele in 25 of 35 cases. In contrast, T-cells sensitized with autologous EBVBLCL alone consistently failed to lyse the same WT1+ leukemia targets.
  • TABLE 3b
    Leukemocidal activity of defined epitope-specific and HLA restricted
    T cells from normal donors sensitized with either autologous EBV BLCL or EBV BLCL loaded
    with pooled WT1 peptides against primary WT1+ leukemia sharing the T cells restricting HLA
    alleles.
    15-mer
    number Cytotoxic CTL response,
    Containing %(at 50:1) E:T ratio vs
    the Presenting WT1+ leukemia expressing
    dominant HLA restricting HLA allele
    epitope Sequence identified allele WT1 CTL EBV CTL
    #1 (−125)-(−117)RQRPHPGAL B0702 67 1
    SEQ ID NO: 142
    #2 (−119)-(−111) GALRNPTAC B0702 60 2
    SEQ ID NO: 143
    #4 (−110)-(−102)PLPHFPPSL A0201 22 3
    SEQ ID NO: 144
    #7 (−99)-(−91)THSPTHPPR B4001 65 0
    SEQ ID NO: 146 A0201 38 3
    #13 (−75)-(67) AILDFLLLQ A0201 19 5
    SEQ ID NO: 142
    #20 (−47)-(39) PGCLQQPEQ A0201 19 10
    SEQ ID NO: 1148
    #24-25 (−27)-(−19)KLGAAEASA A0201 37 5
    SEQ ID NO: 150
    #29-30 (−8)-(−1)ASGSEPQQM B3501 39 0
    SEQ ID NO: 151
    #33 6-15RDLNALLPAV** A0201 9 0
    SEQ ID NO: 152
    #37 22-31 GGCALPVSGA A0201 47 3
    SEQ ID NO: 153
    #41 38-46 LDFAPPGAS A0201 40 0
    SEQ ID NO: 1554
    38-48LDFAPPGASAY DRB10402 40 0
    SEQ ID NO: 156
    #43 46-54SAYGSLGGP* A0201 0 0
    SEQ ID NO: 157 B4001 68 3
    #46 58-66PAPPPPPPP* A0201 0 0
    SEQ ID NO: 158
    #62-63 126-134RMFPNAPYL* A0201 25 2
    SEQ ID NO: 161
    #74-75 174-182HSFKHEDPM B3501 45 5
    SEQ ID NO: 165
    #83-84 209-217CTGSQALLL A0101 33 3
    SEQ ID NO: 167
    #83 206-214TDSCTGSQA B4402 56 1
    SEQ ID NO: 168
    #86 218-226KTPYSSDNL B3503 48 4
    SEQ ID NO: 169 C0401 48 4
    #87 225-233NLYQMTSQLE* A0201 0 0
    SEQ ID NO: 170
    #91 238-246 WNQMNLGAT A0201 19 1
    SEQ ID NO: 171 C1701 16 1
    B3508 19 4
    #91-92 239-248 NQMNLGATL A2402 17 1
    SEQ ID NO: 172
    #91 238-248WNQMNLGATLK DRB11104 0 0
    SEQ ID NO: 173
    235-249CMTWNQMNLGATL DRB10402 17 1
    KG
    SEQ ID NO: 174
    #92 242-250NLGATLKGV A0201 19 2
    SEQ ID NO: 175
    #99-100 269-278GYESDNHTT A0101 33 0
    SEQ ID NO: 178
    #112-113 323-332FMCAYPGCNK B3501 45 5
    SEQ ID NO: 179
    320-334KRPFMCAYPGC DBB10401 5 5
    SEQ ID NO: 180
    #141 436-445NMHQRNHTKL* A0201 0 0
    SEQ ID NO: 183 B4001 72 3
    A2402 0 0
    p < 0.001
    * - the epitope previously predicted by the computer algorithm or described in the literature
    • Example 5 Immunogenicity of the Newly Identified WT1 Epitopes
  • In order to ascertain that that the peptides identified by mapping responses in single donors were also immunogenic in a high proportion of individuals bearing the same presenting HLA allele, it was determined whether these epitopes could elicit appropriately restricted T-cell responses in groups of 6-12 individuals expressing that HLA allele. For this purpose, the T-cells from each donor were sensitized with the identified epitope loaded on a panel of artificial antigen presenting cells (AAPC) (42) each expressing a single HLA allele, specifically A0201, A0301, A2402 or B0702. As shown in Table 4, of 9 peptides identified that are presented by HLA-A0201, all were able to stimulate WT1-specific IFNγ+ T-cell responses in a proportion of HLA-A0201+ individuals. The previously reported 126-134RMFPNAPYL peptide presented by HLA-A0201 allele elicited responses in 5/12 (42%) HLA-A0201+ normal donors tested. In comparison, 5 of the other 8 peptides tested elicited WT1 peptide-specific responses in 50-75% of the same HLA-A0201+ donors. Two WT1 epitopes presented by the HLA-B0702 allele also elicited WT1 specific T-cell responses in 50% and 63% of the tested individuals respectively (Table 4). All of the peptides tested elicited specific responses in at least 2 additional donors bearing their presenting HLA allele.
  • TABLE 4
    Proportion of normal donors responding to identified WT1 peptides
    loaded on AAPCs expressing a single HLA allele.
    WT1 sequence
    predicted
    Sequence Identified Proportion to be
    previously in # of of normal immunogenic Proportion
    identified donors donors when of
    HLA to be after responding presented responses
    allele presented  total to the by the in normal
    ex- by the pool peptide Predicted HLA alleles Predicted donors to
    pressed HLA allele stimulation loaded on Bind- Dissoci- expressed by Bind- Dissoci- the
    by expressed by on AAPCs ing ation the AAPC ing ation peptide
    AAPC the AAPC CAMs (%) index time index time stimulation
    A0201 (−99)-(−91) 1 6/12 3 0 (−99)-(−91) 3 0 6/12
    THSPTHPPR (50%) THSPTHPPR (50%)
    SEQ ID NO: 146 SEQ ID NO: 146
    (−75)-(−67) 1 8/12 19 0.272 (−78)-(−70) 28 225 8/12
    AILDFLLLQ (67%) LLAAILDFL (67%)
    SEQ ID NO: 147 SEQ ID NO: 184
    (−47)-(−39) 2 2/12 0 0 (−45)-(−36) 21 70 2/12
    PGCLQQPEQ (16%) CLQQPEQQGV (16%)
    SEQ ID NO: 148 SEQ ID NO: 185
    (−27)-(−19) 1 8/12 19 17 (−27)-(−19) 19 17 8/12
    KLGAAEASA (67%) KLGAAEASA (67%)
    SEQ ID NO: 149 SEQ ID NO: 150
    6-15 1 3/12 18 0.2 7-15 27 12 3/12
    RDLNALLPAV (25%) DLNALLPAV (25%)
    SEQ ID NO: 152 SEQ ID NO: 186
    10-18 33 181 3/12
    ALLPAVPSL (25%)
    SEQ ID NO: 187
    22-31 3 9/12 13 0.003 22-31 13 0.003 9/12
    GGCALPVSGA (75%) GGCALPVSGA (75%)
    SEQ ID NO: 153 SEQ ID NO: 153
    38-46 2 8/12 11 0 37-45 16 4 0/12
    LDFAPPGAS (67%) VLDFAPPGA (0%)
    SEQ ID NO: 155 SEQ ID NO: 188
    126-134 1 5/12 22 313 126-134 22 313 5/12
    RMFPNAPYL (42%) RMFPNAPYL (42%)
    SEQ ID NO: 161 SEQ ID NO: 161
    238-246 2 3/12 19 0.3 235-243 17 1.5 0/8
    WNQMNLGAT (25%) CMTWNQMNL
    SEQ ID NO: 171 SEQ ID NO: 189
    total pool 13/27 8/12
    (48%) (67%)
    A0301 126-134 1 2/8 10 4.5 124-133 14 0.001 0/8
    RMFPNAPYL (25%) QARMFPNAPY
    SEQ ID NO: 161 SEQ ID NO: 190
    total pool 1/8 2/8
    (12%) (25%)
    A2402 239-248 1 4/6 10 7.2 235-243 10 4 1/6
    NQMNLGATL (60%) CMTWNQMNL (17%)
    SEQ ID NO: 172 SEQ ID NO: 189
    total pool 2/6 6/6
    (33%) (100%)
    B0702 (−125)-(−117) 1 4/8 15 40 (−125)-(−117) 15 40 4/8
    RQRPHPGAL (50%) RQRPHPGAL (50%)
    SEQ ID NO: 142 SEQ ID NO: 142
    (−119)-(−111) 1 5/8 2 0.3 (−118)-(−109) 15 120 5/8
    GALRNPTAC (63%) ALRNPTACPL (63%)
    SEQ ID NO: 143 SEQ ID NO: 191
    323-332 1 3/8 1 0.015 327-335 17 0.4 4/8
    FMCAYPGCNK (38%) YPGCNKRYF (50%)
    SEQ ID NO: 179 SEQ ID NO: 192
    Total pool 2/8 3/8
    (25%) (38%)
    DRB10402 38-48 1 0/2 NA NA 35-49 20 NA ND
    LDFAPPGASAY (tested on APVLDFAPPGAS
    SEQ ID NO: 156 CAMs not AYG
    on AAPC) SEQ ID NO: 193
    • Example 6 Comparison of Responses to Peptides Identified by Mapping Responses to Pooled WT1 15-Mers with Responses to Previously Reported WT1 Peptides Predicted by Binding Algorithms to be Immunogenic
  • Primary responses by normal donor T-cells were compared to individual WT1 peptides identified by the mapping strategy to responses against other WT1 peptides containing flanking sequences predicted to have a higher binding index for the presenting HLA allele using binding algorithms previously described (44,45). As shown in Table 4 above, the predicted binding indices for 8/12 mapped epitopes were only somewhat lower than those for the most studied WT1 peptide, RMF, presented by HLA A0201. However, their dissociation times were markedly lower. Nevertheless, T-cell responses to each of these peptides were elicited in a high proportion of normal donors.
  • In five instances, the mapped peptide specificity (i.e. (−99)-(−91)THS, (−22)-(−19)KLG, 22-31GGC, 126-134RMF and (−125)-(−117)RQR) was identical to the peptide with the highest affinity for the presenting HLA allele predicted by the binding algorithm within the stimulating 15-mer. In those instances in which the mapped sequences and the sequences predicted to have the highest binding index differed, the proportion of donors responding to individual mapped peptides were equal or greater than those generated in response to the neighboring epitopes predicted to have higher affinity. For example, IFNγ+ T-cell responses were generated to the 38-46LDF peptide in 8/12 (67%) of HLA A0201 donors tested, while none responded to the predicted and previously reported (46) epitope 37-45VLDFAPPGA Similarly, among HLA A2402+ donors, 4/6 donors (60%) responded to the 239-248NQMNLGATL peptide while only ⅙ responded to the 235-243 CMTWNQMNL peptide previously reported to be presented by this allele (29).
  • To directly compare peptides presented by HLA A0201 that were identified by matrix mapping with flanking peptides with higher predicted binding indices, the peptides, mixed at equal concentration, were loaded on HLA A0201+ AAPCs and used to sensitize T-cells from 8 of the HLA A0201+ normal donors. After 35 days of sensitization, the T-cells were then washed and secondarily restimulated for 24 hours with aliquots of irradiated autologous PBMC loaded with each individual peptide. Responding IFNγ+ T-cells were then quantitated by FACS. The results, presented in FIG. 5, demonstrate that although the 22-31GGC peptide has the lowest binding index and the shortest predicted dissociation time, it induced strong IFNγ+ T-cell responses in ⅞ donors. Furthermore, although ⅜ donors responded to the 6-15RDL, 10-18 ALL and 7-15 DLN peptides, 6-15 RDL peptides identified by response mapping elicited higher numbers of IFNγ+ T-cells. In comparisons of the (−75)-(−67)AILDFLLLQ with flanking (−78)-(−70)LLAAILDFL sequence, the AIL peptide elicited superior responses and in a higher proportion of donors ( 6/8 vs. ⅜ donors). Similarly, in comparisons of the mapped 38-46LDFAPPGAS peptide with the previously reported 37-45VLDFAPPGA peptide (46) the LDF peptide induced strong responses in 5 of the 8 donors while the VLD peptide induced low responses in only 2 of these donors.
  • Detailed description of FIG. 5. IFNγ+ T-cell responses to equimolar mixtures of 9-mer peptides identified by epitope mapping of in vitro responses and peptides within the same 15-mer or adjacent overlapping 15-mer peptides predicted to have higher binding affinity and immunogenicity. A. Responses to a mixture of nonamers spanning amino acids +2 to +31 including the 6-15 RDL and 22-31GGC peptides to which HLA A0201+ donors responded in epitope mapping studies. B. Responses to the in vitro mapped (−75)-(−67)AILDFLLLQ epitope and a flanking peptide (−78)-(−70)LLAAILDFL with higher predicted binding affinity. C. Responses to the in vitro mapped 38-46LDFAPPGAS epitope and the overlapping 37-45VLDFAPPGA predicted to have higher binding affinity.
  • FIG. 5 presents maps of the WT1 protein. FIG. 5C defines the localization of all previously reported antigenic epitopes presented by HLA class I and II alleles; FIG. 5D depicts the location of immunogenic peptides identified in this report. As can be seen, the 11 epitopes previously reported to be presented by class I and 10 presented by class II HLA alleles are principally clustered in sequences encoded by exons 1, 7 and 10, while the epitopes recognized by normal T-cells sensitized with the WT1 peptide pool are principally clustered in sequences encoded by the first 5 exons. Thus, 26 of the new epitopes are included in each of the four major isoforms of WT1 resulting from splice variants that do or do not include the 17 amino acid sequence (aas 250-266) in exon 5 or the three amino acid sequence (400-410KTS) between zinc fingers 3 and 4. While the epitopes are broadly distributed, clusters of epitopes were detected in the RNA recognition domain in exon 1 and the activation domain (aa 181-250) (FIG. 5F) proximal to the spliced 17aa segment in exon 5. The latter area also contained those epitopes most frequently recognized by multiple donors (FIG. 5E). Nine newly identified epitopes map to a 126 amino acid sequence at the N terminus encoded by a segment of the WT1 gene initially described by Gessler et a137 that is centromeric to exon 1 of the (Exon 5+, KTS+) isoform of WT1 and includes the long isoform of WT1 initiated at a CUG codon upstream of the AUG initiator for exon 1.50 Strikingly, each of the epitopes identified in this sequence elicits IFNγ+ T-cells that are cytolytic against leukemic blasts coexpressing WT1 and the T-cells' restricting HLA allele.
    • Example 7 Inhibitory Effect of Peptides in Ovarian Carcinoma
  • The utility of peptides described herein in treating ovarian cancer was evaluated in two studies. In the first study, the inhibitory effect on ovarian tumor engraftment of T-cells specific for different WT1 peptides was evaluated by pre-incubating T cells at different doses with SKOV3-A2 ovarian carcinoma cells before injection into NOD/SCID mice. T cell cultures specific for the following immunodominant epitopes were prepared using methods described above: A0201 restricted WT1 peptide LKTHTRTHT (SEQ ID NO:182) specific T cells; A0301 restricted WT1 peptide RQRPHPGAL (SEQ ID NO:142) specific T cells, and A0201 restricted WT1 peptide HFPPSLPPT (SEQ ID NO:145) T cells. T cells to tumor cell ratios tested were 50:1, 10:1, 5:1 and control (no T cells). Following tumor injection, the tumor burden was monitored by bioluminescent imaging. For all three T cell lines at each dose, a significant reduction in tumor burden was observed over time vs. control. Furthermore, mouse survival was prolonged by pre-incubation of tumor cells with WT1 peptide specific T cells. In control groups, all mice were dead by 70 days post tumor injection. Increased survival was seen dose-responsively with the T cell: tumor cell dose, which for the 50:1 dose for all T cell lines still had some animals alive at 96 days, and also at 10:1 for the LKTHTRTHT (SEQ ID NO:182) specific line.
  • In a second experiment, WT1 peptide specific T cells were administered intravenously to NOD/SCID mice bearing pre-established ovarian carcinoma SKOV3-A2 xenografts. T cell lines evaluated were: A0201 restricted WT1 peptide LKTHTRTHT (SEQ ID NO:182) specific T cells and A0301 restricted WT1 peptide RQRPHPGAL (SEQ ID NO:142) specific T cells. Tumor burden was monitored by bioluminescence, tumor infiltration by human CD3+ cells evaluated and survival recorded. In both cases, the WT1 specific T cells afforded reduced tumor burden vs. control, increased tumor infiltration by human CD3+ cells and increased survival.
    • Example 8 Recognition of Epitopes by Leukemia Patient T Cells
  • A phase I clinical trial was conducted using transplant donor-derived T-cells sensitized with the full pool of WT1 derived pentadecapeptides described above, in the adoptive therapy of patients who have relapsed following an allogeneic marrow transplant from a normal related or unrelated donor. The HLA restricting alleles and corresponding immunodominant WT1 epitopes are as follows: A0201, SEQ ID NO:147; A0203, SEQ ID NOs:176 and 183; B3503 and C0401, SEQ ID NOs:161 and 169; A6901, SEQ ID NO:177; A0201, SEQ ID NO:182; B4701 and DRB1 0102, SEQ ID NOs: 148 and 149; A3101, SEQ ID NO:145; B4402, SEQ ID NOs:158 and 166; B3503, SEQ ID NOs:146 and 162; DRB 1 1104, SEQ ID NO:149. It is noted that several of the immunodominant epitopes eliciting the WT1 specific T-cells that were used were directed against epitopes in the N-terminal region of the gene, upstream from exon 1, i.e., SEQ ID NOs:145, 147, 148 and 149. Two of the donors responded to PGCLQQPEQQG, SEQ ID NO:149, and both treated patients had temporary clearance of WT1+ leukemic cells following adoptive transfer.
    • Example 9 Pentadecapeptides
  • The following pentadecapeptides were synthesized. H2N refers to the N-terminal end of the peptide, and —COOH the C-terminus.
  • TABLE 5
    Sequence of pentadecapeptides
    SEQ ID NO: 1.H2N-SRQRP HPGAL RNPTA -COOH
    SEQ ID NO: 2.H2N-PHPGA LRNPT ACPLP -COOH
    SEQ ID NO: 3.H2N-ALRNP TACPL PHFPP -COOH
    SEQ ID NO: 4.H2N-PTACP LPHFP PSLPP -COOH
    SEQ ID NO: 5.H2N-PLPHF PPSLP PTHSP -COOH
    SEQ ID NO: 6.H2N-FPPSL PPTHS PTHPP -COOH
    SEQ ID NO: 7.H2N-LPPTH SPTHP PRAGT -COOH
    SEQ ID NO: 8.H2N-HSPTH PPRAG TAAQA -COOH
    SEQ ID NO: 9.H2N-HPPRA GTAAQ APGPR -COOH
    SEQ ID NO: 10.H2N-AGTAA QAPGP RRLLA-COOH
    SEQ ID NO: 11.H2N-AQAPG PRRLL AAILD-COOH
    SEQ ID NO: 12.H2N-GPRRL LAAIL DFLLL-COOH
    SEQ ID NO: 13.H2N-LLAAI LDFLL LQDPA -COOH
    SEQ ID NO: 14.H2N-ILDFL LLQDP ASTCV -COOH
    SEQ ID NO: 15.H2N-LLLQD PASTC VPEPA -COOH
    SEQ ID NO: 16.H2N-DPAST CVPEP ASQHT -COOH
    SEQ ID NO: 17.H2N-TCVPE PASQH TLRSG -COOH
    SEQ ID NO: 18.H2N-EPASQ HTLRS GPGCL -COOH
    SEQ ID NO: 19.H2N-QHTLR SGPGC LQQPE -COOH
    SEQ ID NO: 20.H2N-RSGPG CLQQP EQQGV -COOH
    SEQ ID NO: 21.H2N-GCLQQ PEQQG VRDPG -COOH
    SEQ ID NO: 22.H2N-QPEQQ GVRDP GGIWA -COOH
    SEQ ID NO: 23.H2N-QGVRD PGGIW AKLGA -COOH
    SEQ ID NO: 24.H2N-DPGGI WAKLG AAEAS -COOH
    SEQ ID NO: 25.H2N-IWAKL GAAEA SAERL -COOH
    SEQ ID NO: 26.H2N-LGAAE ASAER LQGRR -COOH
    SEQ ID NO: 27.H2N-EASAE RLQGR RSRGA -COOH
    SEQ ID NO: 28.H2N-ERLQG RRSRG ASGSE -COOH
    SEQ ID NO: 29.H2N-GRRSR GASGS EPQQM -COOH
    SEQ ID NO: 30.H2N-RGASG SEPQQ MGSDV -COOH
    SEQ ID NO: 31.H2N-GSEPQ QMGSD VRDLN -COOH
    SEQ ID NO: 32.H2N-QQMGS DVRDL NALLP -COOH
    SEQ ID NO: 33.H2N-SDVRD LNALL PAVPS -COOH
    SEQ ID NO: 34.H2N-DLNAL LPAVP SLGGG -COOH
    SEQ ID NO: 35.H2N-LLPAV PSLGG GGGCA -COOH
    SEQ ID NO: 36.H2N-VPSLG GGGGC ALPVS -COOH
    SEQ ID NO: 37.H2N-GGGGG CALPV SGAAQ -COOH
    SEQ ID NO: 38.H2N-GCALP VSGAA QWAPV -COOH
    SEQ ID NO: 39.H2N-PVSGA AQWAP VLDFA -COOH
    SEQ ID NO: 40.H2N-AAQWA PVLDF APPGA -COOH
    SEQ ID NO: 41.H2N-APVLD FAPPG ASAYG -COOH
    SEQ ID NO: 42.H2N-DFAPP GASAY GSLGG -COOH
    SEQ ID NO: 43.H2N-PGASA YGSLG GPAPP -COOH
    SEQ ID NO: 44.H2N-AYGSL GGPAP PPAPP -COOH
    SEQ ID NO: 45.H2N-LGGPA PPPAP PPPPP -COOH
    SEQ ID NO: 46.H2N-APPPA PPPPP PPPPH -COOH
    SEQ ID NO: 47.H2N-APPPP PPPPP HSFIK -COOH
    SEQ ID NO: 48.H2N-PPPPP PHSFI KQEPS -COOH
    SEQ ID NO: 49.H2N-PPHSF IKQEP SWGGA -COOH
    SEQ ID NO: 50.H2N-FIKQE PSWGG AEPHE -COOH
    SEQ ID NO: 51.H2N-EPSWG GAEPH EEQCL -COOH
    SEQ ID NO: 52.H2N-GGAEP HEEQC LSAFT -COOH
    SEQ ID NO: 53.H2N-PHEEQ CLSAF TVHFS -COOH
    SEQ ID NO: 54.H2N-QCLSA FTVHF SGQFT -COOH
    SEQ ID NO: 55.H2N-AFTVH FSGQF TGTAG -COOH
    SEQ ID NO: 56.H2N-HFSGQ FTGTA GACRY -COOH
    SEQ ID NO: 57.H2N-QFTGT AGACR YGPFG -COOH
    SEQ ID NO: 58.H2N-TAGAC RYGPF GPPPP -COOH
    SEQ ID NO: 59.H2N-CRYGP FGPPP PSQAS -COOH
    SEQ ID NO: 60.H2N-PFGPP PPSQA SSGQA -COOH
    SEQ ID NO: 61.H2N-PPPSQ ASSGQ ARMFP -COOH
    SEQ ID NO: 62.H2N-QASSG QARMF PNAPY -COOH
    SEQ ID NO: 63.H2N-GQARM FPNAP YLPSC -COOH
    SEQ ID NO: 64.H2N-MFPNA PYLPS CLESQ -COOH
    SEQ ID NO: 65.H2N-APYLP SCLES QPAIR -COOH
    SEQ ID NO: 66.H2N-PSCLE SQPAI RNQGY -COOH
    SEQ ID NO: 67.H2N-ESQPA IRNQG YSTVT -COOH
    SEQ ID NO: 68.H2N-AIRNQ GYSTV TFDGT -COOH
    SEQ ID NO: 69.H2N-QGYST VTFDG TPSYG -COOH
    SEQ ID NO: 70.H2N-TVTFD GTPSY GHTPS -COOH
    SEQ ID NO: 71.H2N-DGTPS YGHTP SHHAA -COOH
    SEQ ID NO: 72.H2N-SYGHT PSHHA AQFPN -COOH
    SEQ ID NO: 73.H2N-TPSHH AAQFP NHSFK -COOH
    SEQ ID NO: 74.H2N-HAAQF PNHSF KHEDP -COOH
    SEQ ID NO: 75.H2N-FPNHS FKHED PMGQQ -COOH
    SEQ ID NO: 76.H2N-SFKHE DPMGQ QGSLG -COOH
    SEQ ID NO: 77.H2N-EDPMG QQGSL GEQQY -COOH
    SEQ ID NO: 78.H2N-GQQGS LGEQQ YSVPP -COOH
    SEQ ID NO: 79.H2N-SLGEQ QYSVP PPVYG-COOH
    SEQ ID NO: 80.H2N-QQYSV PPPVY GCHTP-COOH
    SEQ ID NO: 81.H2N-VPPPV YGCHT PTDSC -COOH
    SEQ ID NO: 82.H2N-VYGCH TPTDS CTGSQ -COOH
    SEQ ID NO: 83.H2N-HTPTD SCTGS QALLL -COOH
    SEQ ID NO: 84.H2N-DSCTG SQALL LRTPY -COOH
    SEQ ID NO: 85.H2N-GSQAL LLRTP YSSDN -COOH
    SEQ ID NO: 86.H2N-LLLRT PYSSD NLYQM -COOH
    SEQ ID NO: 87.H2N-TPYSS DNLYQ MTSQL -COOH
    SEQ ID NO: 88.H2N-SDNLY QMTSQ LECMT -COOH
    SEQ ID NO: 89.H2N-YQMTS QLECM TWNQM -COOH
    SEQ ID NO: 90.H2N-SQLEC MTWNQ MNLGA -COOH
    SEQ ID NO: 91.H2N-CMTWN QMNLG ATLKG -COOH
    SEQ ID NO: 92.H2N-NQMNL GATLK GVAAG -COOH
    SEQ ID NO: 93.H2N-LGATL KGVAA GSSSS -COOH
    SEQ ID NO: 94.H2N-LKGVA AGSSS SVKWT -COOH
    SEQ ID NO: 95.H2N-AAGSS SSVKW TEGQS -COOH
    SEQ ID NO: 96.H2N-SSSVK WTEGQ SNHST -COOH
    SEQ ID NO: 97.H2N-KWTEG QSNHS TGYES -COOH
    SEQ ID NO: 98.H2N-GQSNH STGYE SDNHT -COOH
    SEQ ID NO: 99.H2N-HSTGY ESDNH TTPIL -COOH
    SEQ ID NO: 100.H2N-YESDN HTTPI LCGAQ -COOH
    SEQ ID NO: 101.H2N-NHTTP ILCGA QYRIH -COOH
    SEQ ID NO: 102.H2N-PILCG AQYRI HTHGV -COOH
    SEQ ID NO: 103.H2N-GAQYR IHTHG VFRGI -COOH
    SEQ ID NO: 104.H2N-RIHTH GVFRG IQDVR -COOH
    SEQ ID NO: 105.H2N-HGVFR GIQDV RRVPG -COOH
    SEQ ID NO: 106.H2N-RGIQD VRRVP GVAPT -COOH
    SEQ ID NO: 107.H2N-DVRRV PGVAP TLVRS -COOH
    SEQ ID NO: 108.H2N-VPGVA PTLVR SASET -COOH
    SEQ ID NO: 109.H2N-APTLV RSASE TSEKR -COOH
    SEQ ID NO: 110.H2N-VRSAS ETSEK RPFMC -COOH
    SEQ ID NO: 111.H2N-SETSE KRPFM CAYPG -COOH
    SEQ ID NO: 112.H2N-EKRPF MCAYP GCNKR -COOH
    SEQ ID NO: 113.H2N-FMCAY PGCNK RYFKL -COOH
    SEQ ID NO: 114.H2N-YPGCN KRYFK LSHLQ -COOH
    SEQ ID NO: 115.H2N-NKRYF KLSHL QMHSR -COOH
    SEQ ID NO: 116.H2N-FKLSH LQMHS RKHTG -COOH
    SEQ ID NO: 117.H2N-HLQMH SRKHT GEKPY -COOH
    SEQ ID NO: 118.H2N-HSRKH TGEKP YQCDF -COOH
    SEQ ID NO: 119.H2N-HTGEK PYQCD FKDCE -COOH
    SEQ ID NO: 120.H2N-KPYQC DFKDC ERRFS -COOH
    SEQ ID NO: 121.H2N-CDFKD CERRF SRSDQ-COOH
    SEQ ID NO: 122.H2N-DCERR FSRSD QLKRH-COOH
    SEQ ID NO: 123 H2N-RFSRS DQLKR HQRRH-COOH
    SEQ ID NO: 124.H2N-SDQLK RHQRR HTGVK-COOH
    SEQ ID NO: 125.H2N-KRHQR RHTGV KPFQC-COOH
    SEQ ID NO: 126.H2N-RRHTG VKPFQ CKTCQ-COOH
    SEQ ID NO: 127.H2N-GVKPF QCKTC QRKFS-COOH
    SEQ ID NO: 128.H2N-FQCKT CQRKF SRSDH-COOH
    SEQ ID NO: 129.H2N-TCQRK FSRSD HLKTH-COOH
    SEQ ID NO: 130.H2N-KFSRS DHLKT HTRTH-COOH
    SEQ ID NO: 131.H2N-SDHLK THTRT HTGKT-COOH
    SEQ ID NO: 132.H2N-KTHTR THTGK TSEKP-COOH
    SEQ ID NO: 133.H2N-RTHTG KTSEK PFSCR-COOH
    SEQ ID NO: 134.H2N-GKTSE KPFSC RWPSC-COOH
    SEQ ID NO: 135.H2N-EKPFS CRWPS CQKKF-COOH
    SEQ ID NO: 136.H2N-SCRWP SCQKK FARSD-COOH
    SEQ ID NO: 137.H2N-PSCQK KFARS DELVR-COOH
    SEQ ID NO: 138.H2N-KKFAR SDELV RHHNM-COOH
    SEQ ID NO: 139.H2N-RSDEL VRHHN MHQRN-COOH
    SEQ ID NO: 140.H2N-LVRHH NMHQR NMTKL-COOH
    SEQ ID NO: 141.H2N-HNMHQ RNMTK LQLAL-COOH
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Claims (31)

1. An isolated WT-1 peptide consisting of an amino acid sequence selected from among AILDFLLLQ (SEQ ID NO:147), RQRPHPGAL (SEQ ID NO:142), GALRNPTAC (SEQ ID NO:143), THSPTHPPR (SEQ ID NO:146), WNQMNLGATLK (SEQ ID NO:173), PGCLQQPEQQG (SEQ ID NO:149), LDFAPPGASAY (SEQ ID NO:156), PLPHFPPSL (SEQ ID NO:144), HFPPSLPPT (SEQ ID NO:145), LLAAILDFL (SEQ ID NO:184), CLQQPEQQGV (SEQ ID NO:185), ALRNPTACPL (SEQ ID NO:191), GGCALPVSGA (SEQ ID NO:153), LGATLKGVAA (SEQ ID NO:176), TLGVAAGS (SEQ ID NO:177), KRPFMCAYPGC (SEQ ID NO:180) LKTHTRTHT (SEQ ID NO:182) and SEQ ID NOS:1-15.
2-4. (canceled)
5. An isolated WT-1 peptide consisting of 8-30 amino acids comprising an amino acid sequence selected from SEQ ID NO: 142, 143, 144, 145, 146, 147, 149 and 184.
6. An isolated WT-1 peptide consisting of 16-30 amino acids comprising an amino acid sequence selected from SEQ ID NO:1-15.
7. The isolated WT-1 peptide of claim 1 or 5, wherein said isolated WT-1 peptide can bind to an HLA class I molecule, an HLA class II molecule, or the combination thereof.
8. A pharmaceutical composition comprising one or more peptides of claim 1 or 5 and a pharmaceutically acceptable carrier, vehicle or excipient.
9. A vaccine comprising (a) one or more isolated WT-1 peptides of claim 1 or 5 and (b) an adjuvant or a carrier.
10. The vaccine of claim 9, wherein said adjuvant is QS21, Freund's incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG, alum, a growth factor, a cytokine, a chemokine, an interleukin, Montanide or GM-CSF.
11. A method of treating a subject with a WT-1-expressing cancer or reducing an incidence of a WT-1-expressing cancer, or its relapse, the method comprising administering to said subject one or more peptides of any one of claim 1, 5 or 6, a composition thereof, a composition thereof further comprising an antigen presenting cell, or a vaccine thereof or any combination thereof, thereby treating a subject with a WT-1-expressing cancer, reducing an incidence of a WT-1-expressing cancer or its relapse therein.
12. The method of claim 11, wherein said WT-1-expressing cancer is a leukemia, a desmoplastic small round cell tumor, a gastric cancer, a colon cancer, a lung cancer, a breast cancer, a germ cell tumor, an ovarian cancer, a uterine cancer, a thyroid cancer, a liver cancer, a renal cancer, a Kaposi's sarcoma, a sarcoma, a hepatocellular carcinoma, a Wilms' tumor, an acute myelogenous leukemia (AML), a myelodysplastic syndrome (MDS), mesothelioma or a non-small cell lung cancer (NSCLC).
13. A method of inducing the formation and proliferation of CTL specific for cells of a WT-1-expressing cancer, the method comprising administering to said subject one or more peptides of any one of claim 1, 5 or 6, a composition thereof or a vaccine thereof or any combination thereof, thereby inducing the formation and proliferation of CTL specific for cells of a WT-1-expressing cancer.
14. The method of claim 13, wherein said WT-1-expressing cancer is a leukemia, a desmoplastic small round cell tumor, a gastric cancer, a colon cancer, a lung cancer, a breast cancer, a germ cell tumor, an ovarian cancer, a uterine cancer, a thyroid cancer, a liver cancer, a renal cancer, a kaposi's sarcoma, a sarcoma, a hepatocellular carcinoma, a Wilms' tumor, an acute myelogenous leukemia (AML), a myelodysplastic syndrome (MDS), mesothelioma or a non-small cell lung cancer (NSCLC).
15. A composition comprising (a) an antigen-presenting cell and (b) one or more peptides of claim 1 or 5.
16. The vaccine of claim 9 further comprising a cell population xx.
17. The composition of claim 15 wherein the antigen-presenting cell is a dendritic cell, monocyte, macrophage, cytokine-activated monocyte or an EBV-transformed B-lymphoblastoid cell.
18. The composition of claim 15 wherein the antigen-presenting cell is from a cell line.
19. The vaccine of claim 16 wherein the cell population is selected from lymphocytes, monocytes, macrophages, dendritic cells, endothelial cells, stem cells or any combination thereof.
20. The vaccine of claim 16 wherein the cell population is autologous, syngeneic or allogeneic.
21. The vaccine of claim 16 wherein the population is obtained from peripheral blood, leukopheresis blood product, apheresis blood product, peripheral lymph nodes, gut associated lymphoid tissue, spleen, thymus, cord blood, mesenteric lymph nodes, liver, a site of immunologic lesions, pancreas, cerebrospinal fluid, a tumor sample, or granulomatous tissue.
22. A method of inducing formation and proliferation of WT1 protein-specific cytotoxic T lymphocytes comprising contacting a lymphocyte population in vitro or ex vivo with one or more peptides selected from AILDFLLLQ (SEQ ID NO:147), RQRPHPGAL (SEQ ID NO:142), GALRNPTAC (SEQ ID NO:143), THSPTHPPR (SEQ ID NO:146), PGCLQQPEQQG (SEQ ID NO:149), LDFAPPGASAY (SEQ ID NO:156), PLPHFPPSL (SEQ ID NO:144), HFPPSLPPT (SEQ ID NO:145), LLAAILDFL (SEQ ID NO:184), ALRNPTACPL (SEQ ID NO:191), GGCALPVSGA (SEQ ID NO:153), WNQMNLGATLK (SEQ ID NO:173), LGATLKGVAA (SEQ ID NO:176), TLGVAAGS (SEQ ID NO:177), KRPFMCAYPGC (SEQ ID NO:180), LKTHTRTHT (SEQ ID NO:182), and SEQ ID NOS:1-15; or an isolated WT1 peptide consisting of 8-30 amino acids comprising an amino acid sequence selected from SEQ ID NO: 142, 143, 144, 145, 146, 147, 149, and 184; or an isolated WT1 peptide consisting of 16-30 amino acids comprising an amino acid sequence selected from SEQ ID NOS:1-15, a composition thereof, a composition thereof further comprising an antigen presenting cell, or a vaccine thereof, or any combination thereof, thereby inducing formation and proliferation of WT1 protein-specific cytotoxic T lymphocytes.
23. The method of claim 22 wherein the peptide is a pool of peptides having SEQ ID NOS:1-141.
24. A method of treating a subject with a WT1-expressing cancer or reducing an incidence of a WT1-expressing cancer, or its relapse, the method comprising administering to said subject WT1 protein-specific cytotoxic T lymphocytes obtained by the method of claim 22, thereby treating a subject with a WT1-expressing cancer, reducing an incidence of a WT1-expressing cancer or its relapse therein.
25. The method of claim 24 wherein the peptide is a pool of peptides having SEQ ID NOS:1-141.
26. A method of inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein, or the combination thereof, the method comprising contacting a lymphocyte population in vitro or ex vivo with one or more peptides selected from AILDFLLLQ (SEQ ID NO:147), RQRPHPGAL (SEQ ID NO:142), GALRNPTAC (SEQ ID NO:143), THSPTHPPR (SEQ ID NO:146), PGCLQQPEQQG (SEQ ID NO:149), LDFAPPGASAY (SEQ ID NO:156), PLPHFPPSL (SEQ ID NO:144), HFPPSLPPT (SEQ ID NO:145), LLAAILDFL (SEQ ID NO:184), ALRNPTACPL (SEQ ID NO:191), GGCALPVSGA (SEQ ID NO:153), WNQMNLGATLK (SEQ ID NO:173), LGATLKGVAA (SEQ ID NO:176), TLGVAAGS (SEQ ID NO:177), KRPFMCAYPGC (SEQ ID NO:180), LKTHTRTHT (SEQ ID NO:182) and SEQ ID NOS:1-15; or an isolated WT1 peptide consisting of 8-30 amino acids comprising an amino acid sequence selected from SEQ ID NO: 142, 143, 144, 145, 146, 147, 149, and 184; or an isolated WT1 peptide consisting of 16-30 amino acids comprising an amino acid sequence selected from SEQ ID NOS:1-15, a composition thereof, a composition thereof further comprising an antigen presenting cell, or a vaccine thereof, or any combination thereof, thereby inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein; or a combination thereof.
27. The method of claim 26 wherein the peptides are a pool of peptides having SEQ ID NOS:1-141.
28. The method of any one of claim 22, 24 or 26 wherein the lymphocyte population is obtained from a donor.
29. The method of any one of claim 22, 24 or 26 wherein the lymphocyte population is obtained from a human source.
30. The method of claim 22, 24 or 26, wherein said WT1-expressing cancer is a leukemia, a desmoplastic small round cell tumor, a gastric cancer, a colon cancer, a lung cancer, a breast cancer, a germ cell tumor, an ovarian cancer, a uterine cancer, a thyroid cancer, a liver cancer, a renal cancer, a Kaposi's sarcoma, a sarcoma, a hepatocellular carcinoma, a Wilms' tumor, an acute myelogenous leukemia (AML), a myelodysplastic syndrome (MDS), mesothelioma or a non-small cell lung cancer (NSCLC).
31. A method of inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein, or the combination thereof, the method comprising administering to a subject one or more peptides of any one of claim 1, 5 or 6, a composition thereof, a composition thereof further comprising an antigen presenting cell, or a vaccine thereof, or any combination thereof, thereby inducing formation and proliferation of (a) a WT1 protein-specific CD8+ lymphocyte; or (b) a CD4+ lymphocyte specific for the WT1 protein; or a combination thereof.
32. The method of claim 31, wherein said WT1-expressing cancer is a leukemia, a desmoplastic small round cell tumor, a gastric cancer, a colon cancer, a lung cancer, a breast cancer, a germ cell tumor, an ovarian cancer, a uterine cancer, a thyroid cancer, a liver cancer, a renal cancer, a Kaposi's sarcoma, a sarcoma, a hepatocellular carcinoma, a Wilms' tumor, an acute myelogenous leukemia (AML), a myelodysplastic syndrome (MDS), mesothelioma or a non-small cell lung cancer (NSCLC).
33. A method of treating a subject with a WT1-expressing cancer, the method comprising administering to said subject WT1 protein-specific cytotoxic T lymphocytes obtained by the method of claim 26, thereby treating a subject with a WT1-expressing cancer.
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